Analogs of parathyroid hormone and parathyroid hormone relate peptide synthesis and use for the treatment of osteoporosis

ABSTRACT

Synthetic polypeptide analogs of parathyroid hormone PTH, parathyroid hormone related peptide PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, in which amino acid residues (22-31) form an amphipathic α-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence: 
     
         Haa(Laa Laa Haa Haa).sub.2 Laa 
    
     and their pharmaceutically acceptable salts are useful for the prophylaxis and treatment of ostoporosis in mammals. Processes for the production of the polypeptides via solid phase and recombinant methods are provided.

This is a division of application Ser. No. 08/184,328, filed Jan. 18,1994, which is a continuation-in-part of pending U.S. patent applicationSer. No. 07/915,247, filed Jul. 14, 1992 now U.S. Pat. No. 5,589,452.

BACKGROUND OF THE INVENTION

a) Field of the Invention

This invention relates to novel analogs of parathyroid hormone andparathyroid hormone related peptide, their synthesis by solid phase andrecombinant techniques, and their use for increasing bone mass inmammalian subjects.

b) Description of Related Art

Osteoporosis is the most common form of metabolic bone disease and maybe considered the symptomatic, fracture stage of bone loss (osteopenia).Although osteoporosis may occur secondary to a number of underlyingdiseases, 90% of all cases appear to be idiopathic. Postmenopausal womenare particularly at risk for idiopathic osteoporosis (postmenopausal orType I osteoporosis). Another high risk group for idiopathicosteoporosis is the elderly of either sex (senile or Type IIosteoporosis). Osteoporosis has also been related to corticosteroid use,immobilization or extended bed rest, alcoholism, diabetes, gonadotoxicchemotherapy, hyperprolactinemia, anorexia nervosa, primary andsecondary amenorrhea, and oophorectomy.

In the various forms of osteoporosis, bone fractures, which are theresult of bone loss that has reached the point of mechanical failure,frequently occur. Postmenopausal osteoporosis is characterized byfractures of the wrist and spine, while femoral neck fractures seem tobe the dominant feature of senile osteoporosis.

The mechanism by which bone is lost in osteoporotics is believed toinvolve an imbalance in the process by which the skeleton renews itself.This process has been termed bone remodeling. It occurs in a series ofdiscrete pockets of activity. These pockets appear spontaneously withinthe bone matrix on a given bone surface as a site of bone resorption.Osteoclasts (bone dissolving or resorbing cells) are responsible for theresorption of a portion of bone of generally constant dimension. Thisresorption process is followed by the appearance of osteoblasts (boneforming cells) which then refill with new bone the cavity left by theosteoclasts.

In a healthy adult subject, the rate at which osteoclasts andosteoblasts are formed is such that bone formation and bone resorptionare in balance. However, in osteoporotics an imbalance in the boneremodeling process develops which results in bone being lost at a ratefaster than it is being made. Although this imbalance occurs to someextent in most individuals as they age, it is much more severe andoccurs at a younger age in postmenopausal osteoporotics or followingoophorectomy.

There have been many attempts to treat osteoporosis with the goal ofeither slowing further bone loss or, more desirably, producing a netgain in bone mass. Certain agents, such as estrogen and thebisphosphonates, appear to slow further bone loss in osteoporotics.Agents which slow bone loss, because of the different durations of boneresorption and formation, may appear to increase bone mass (on the orderof 3 to 7%). However, this apparent increase is limited in time, notprogressive, and is due to a decrease in "remodeling space." Inaddition, because of the close coupling between resorption andformation, treatments which impede bone resorption also ultimatelyimpede bone formation.

It has been suggested that treatment with parathyroid hormone (PTH)would lead to both increased bone turnover and a positive calciumbalance. However, human clinical trials have shown that any increase intrabecular bone is offset by a decrease in cortical bone, so that thereis no net increase in total bone.

Hefti, et al. in Clinical Science 62, 389-396 (1982) have reported thatdaily subcutaneous doses of either bPTH(1-84) or hPTH(1-34) increasedwhole body calcium and ash weight of individual bones in both normal andosteoporotic adult female rats.

Liu, et al. in J. Bone Miner. Res. 6:10, 1071-1080 (1991) have notedthat ovariectomy of adult female rats induced a 47% loss in thepercentage of trabecular bone in the proximal tibial metaphysis,accompanied by a significant increase in the number of osteoblasts andtrabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34)completely reversed the loss of trabecular bone and resulted in amountsof trabecular bone exceeding that of sham operated controls. The numberof osteoblasts increased and the number of osteoclasts decreased.

Hock et al. in J. Bone Min. Res. 7:1, 65-71 (1992) have reported thatdaily subcutaneous injections of hPTH(1-34) to healthy adult male ratsfor 12 days increased trabecular and cortical bone calcium and dryweight. Total bone mass, trabecular bone volume, trabecular thicknessand number, and osteoblastic surfaces were increased.

The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH),and porcine (pPTH), are single polypeptide chains of 84 amino acidresidues, with molecular weights of approximately 9500. Biologicalactivity is associated with the N-terminal portion, with residues (1-34)apparently the minimum required.

The N-terminal segment of human PTH differs from the N-terminal segmentof the bovine and porcine hormones by only three and two amino acidresidues, respectively: ##STR1##

The primary function of PTH is to elicit the adaptive changes that serveto maintain a constant concentration of Ca²⁺ in the extracellular fluid.PTH acts on the kidneys to increase tubular reabsorption of Ca²⁺ fromthe urine, as well as stimulating the conversion of calcifediol tocalcitriol, which is responsible for absorption of Ca²⁺ from theintestines. One prominent effect is to promote the mobilization of Ca²⁺from bone. PTH acts on bone to increase the rate of resorption of Ca²⁺and phosphate. PTH stimulates the rate of bone resorption byosteoclasts, increases the rate of differentiation of mesenchymal cellsto osteoclasts, and prolongs the half life of these latter cells. Withprolonged action of PTH the number of bone forming osteoblasts is alsoincreased; thus, the rate of bone turnover and remodeling is enhanced.However, individual osteoblasts appear to be less active than normal.

Rosenblatt, et al. in U.S. Pat. Nos. 4,423,037, 4,968,669 and 5,001,223have disclosed PTH antagonists obtained by the deletion of theN-terminal (1-6) amino acids and the selective replacement of Phe⁷,Met⁸,18, and Gly¹² -Tyr³⁴ -NH₂ reportedly increased the activity andstability of these compounds.

Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein,and fragments thereof, reproduce the major biological actions of PTH.PTHrp is elaborated by a number of human and animal tumors and othertissues and may play a role in hypercalcemia of malignancy. The sequenceof hPTHrp (1-34) is as follows: ##STR2##

The sequence homology between hPTH and hPTHrp is largely limited to the13 N-terminal residues, 8 of which are identical; only 1 of 10 aminoacids in the (25-34) receptor binding region of hPTH is conserved inhPTHrp. Conformational similarity may underlie the common activity.Cohen, et al. in J. Biol. Chem 266:3, 1997-2004 (1991) have suggestedthat much of the sequence of PTH(1-34) and PTHrp(1-34), in particularregions (5-18) and (21-34), assumes an α-helical configuration, whilenoting that there is some question whether this configuration prevailsfor the carboxyl terminal end under physiological conditions. Such asecondary structure may be important for lipid interaction, receptorinteraction, and/or structural stabilization.

We have synthesized analogs of PTH and of PTHrp with the objective ofdeveloping improved therapeutic agents for the restoration of bone massin mammalian subjects, including those afflicted with osteoporosis.

SUMMARY OF THE INVENTION

This invention provides synthetic polypeptide analogs of parathyroidhormone (PTH), parathyroid hormone related peptide (PTHrp), and of thephysiologically active truncated homologs and analogs of PTH and PTHrp,and salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, said residues (22-31) selected from hydrophilicamino acids (Haa) and lipophilic amino acids (Laa) ordered in thesequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

When specific illustrative embodiments of this sequence type areinserted into PTH, PTHrp, and the physiologically active truncatedanalogs and homologs of PTH and PTHrp, the resulting polypeptides areeffective bone remodeling agents.

In one aspect, then, this invention provides analogs of PTH, PTHrp, andthe physiologically active truncated analogs and homologs of PTH andPTHrp, or salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, the sequence of said residues (22-31) selectedfrom: ##STR3## Xaa¹ and Xaa⁴ are independently Glu, Glu(OCH₃), His, orPhe; Xaa² is Leu or Phe; Xaa⁵ is Lys or His; Xaa⁷ and Xaa¹⁰ areindependently Leu or Ile; Xaa8 is Ala, Arg, or Glu; and Xaa⁹ is Lys orGlu (SEQ ID NO:85); preferably ##STR4## wherein Xaa is Glu or Arg (SEQID NO:26); ##STR5## wherein Xaa¹ and Xaa⁴ are independently Glu,Glu(OCH₃), His, or Phe; Xaa² is Leu or Phe; Xaa⁸ is Glu, Lys, orLys(COCH₂ PEGX) and PEGX is a poly-(ethylene glycol methyl ether)radical of molecular weight 100 to 10,000 (SEQ ID NO:86); preferably,##STR6## wherein Xaa is Glu, Lys, or Lys (COCH2PEGX) and PEGX is apoly-(ethylene glycol methyl ether) radical of molecular weight 100 to10,000 (SEQ ID NO:27); ##STR7##

In another aspect, this invention provides analogs of PTH, PTHrp, and ofthe physiologically active truncated homologs and analogs of PTH andPTHrp, or salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, the sequence of said residues (22-31) selectedfrom: ##STR8## wherein Xaa is Glu or Arg (SEQ ID NO:26); ##STR9##wherein Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is a poly-(ethyleneglycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ IDNO:27); ##STR10##

Also provided are pharmaceutical compositions for the prevention ortreatment of conditions characterized by decreases in bone masscomprising an effective bone mass increasing amount of a polypeptideanalog of parathyroid hormone (PTH), parathyroid hormone related peptide(PTHrp), and of the physiologically active truncated homologs andanalogs of PTH and PTHrp, and salts thereof, in which amino acidresidues (22-31) form an amphipathic α-helix, said residues (22-31)selected from hydrophilic amino acids (Haa) and lipophilic amino acids(Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

in admixture with a pharmaceutically acceptable carrier. Also providedare processes for preparing pharmaceutical compositions which comprisemixing the above described compounds with a pharmaceutically acceptablecarrier.

Further, this invention provides methods for treating mammalianconditions characterized by decreases in bone mass which methodscomprise administering to a mammal in need thereof an effective bonemass increasing amount of a polypeptide analog of PTH, PTHrp, or of aphysiologically active truncated homolog or analog of PTH or PTHrp, or asalt thereof, in which amino acid residues (22-31) form an amphipathicα-helix, said residues (22-31) selected from hydrophilic amino acids(Haa) and lipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

More specifically, this invention provides methods for treatingmammalian conditions characterized by decreases in bone mass whichmethods comprise administering to a mammal in need thereof an effectivebone mass increasing amount of a polypeptide analog of PTH, PTHrp, or ofa physiologically active truncated homolog or analog of PTH or PTHrp, orsalt thereof, in which amino acid residues (22-31) form an amphipathicα-helix, the sequence of said residues (22-31) selected from: (SEQ IDNOS: 85, 86, 26, 27, 28, 29, and 30).

This invention also includes processes for the solid phase synthesis ofpolypeptide analogs of PTH, PTHrp, and of the physiologically activetruncated homologs and analogs of PTH and PTHrp, and salts thereof, inwhich amino acid residues (22-31) form an amphipathic α-helix, saidresidues (22-31) selected from hydrophilic amino acids (Haa) andlipophilic amino acids (Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa,

which processes comprise sequentially coupling protected amino acids ona suitable resin support, removing the side chain and N.sup.α-protecting groups, and cleaving the polypeptide from the resin.

This invention also encompasses processes for the solid phase synthesisof polypeptide analogs of PTH, pTHrp, and of the physiologically activetruncated homologs and analogs of PTH and PTHrp, and salts thereof, inwhich amino acid residues (22-31) form an amphipathic α-helix, thesequence of said residues (22-31) selected from: (SEQ ID NOS: 85, 86,26, 27, 28, 29, and 30); which processes comprise sequentially couplingprotected amino acids on a suitable resin support, removing the sidechain and N.sup.α -protecting groups, and cleaving the polypeptide fromthe resin.

Also included are processes for the recombinant synthesis of polypeptideanalogs of PTH, PTHrp, and of the physiologically active truncatedhomologs and analogs of PTH and PTHrp, and salts thereof, in which aminoacid residues (22-31) form an amphipathic α-helix, said residues (22-31)selected from hydrophilic amino acids (Haa) and lipophilic amino acids(Laa) ordered in the sequence:

    Haa (Laa Laa Haa Haa).sub.2 Laa.

The invention also encompasses DNA sequences, vectors, and plasmids forthe recombinant synthesis of such polypeptide analogs. Specifically, theinvention provides DNA sequences, vectors, and plasmids for therecombinant synthesis of polypeptide analogs of PTH, PTHrp, and of thephysiologically active truncated homologs and analogs of PTH and PTHrp,and salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, the sequence of said residues (22-31) selectedfrom: (SEQ ID NOS: 85, 86, 26, 27, 28, 29, and 30).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the DNA sequence and enzyme restriction sites of asynthetic gene coding for a PTHrp(1-34) analog of this invention.

FIG. 2 outlines the preparation of a plasmid incorporating a PTHrp(1-34)analog gene.

FIG. 3 outlines the preparation of a plasmid incorporating two copies ofa PTHrp(1-34) analog gene.

FIG. 4 outlines the preparation of a plasmid incorporating four copiesof a PTHrp(1-34) analog gene.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Definitions

The one- and three-letter abbreviations for the various commonnucleotide bases and amino acids are as recommended in Pure Appl. Chem.31, 639-645 (1972) and 40, 277-290 (1974) and the IUPAC-IUB BiochemicalNomenclature Commission and comply with 37 CFR §1.822 (55 FR 18245, May1, 1990). The one- and three-letter abbreviations are as follows:

    ______________________________________                                        Amino Acid Abbreviations                                                                      Three-letter                                                                            One-letter                                          Amino Acid      Symbol    Symbol                                              ______________________________________                                        Alanine         Ala       A                                                   Arginine        Arg       R                                                   Asparagine      Asn       N                                                   Aspartic Acid   Asp       D                                                   Asn + Asp       Asx       B                                                   Cysteine        Cys       C                                                   Glutamine       Gln       Q                                                   Glutamic Acid   Glu       E                                                   Gln + Glu       Glx       Z                                                   Glycine         Gly       G                                                   Histidine       His       H                                                   Isoleucine      Ile       I                                                   Leucine         Leu       L                                                   Lysine          Lys       K                                                   Methionine      Met       M                                                   Phenylalanine   Phe       F                                                   Proline         Pro       P                                                   Serine          Ser       S                                                   Threonine       Thr       T                                                   Tryptophan      Trp       W                                                   Tyrosine        Tyr       Y                                                   Valine          Val       V                                                   Other amino acid                                                                              Xaa       X                                                   ______________________________________                                    

The abbreviations represent L-amino acids unless otherwise designated asD- or D,L-. Certain amino acids, both natural and non-natural, areachiral, e.g. glycine. All peptide sequences are presented with theN-terminal amino acid on the left and the C-terminal amino acid on theright.

Further abbreviations for other amino acids and compounds used hereinare:

    ______________________________________                                        hSer   homoserine                                                             hSerlac                                                                              homoserine lactone                                                     Nle    norleucine                                                             PEG2   radical of diethylene glycol methyl ether, a.k.a.                             methoxydi(ethyleneoxy), CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.2 --,             (MW = 119)                                                             PEG5000                                                                              radical of poly(ethylene glycol methyl ether), a.k.a.                         methoxypoly(ethyleneoxy), CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.110             --,                                                                           (avg. MW = 5000)                                                       PEGX   radical of poly(ethylene glycol methyl ether),                                CH.sub.3 O(CH.sub.2 CH.sub.2 O).sub.n --, n = 2-225, (avg. MW =               100 to 10,000)                                                         ______________________________________                                    

"Hydrophilic amino acid (Haa)" refers to an amino acid having at leastone hydrophilic functional group in addition to those required forpeptide bond formation, such as arginine, asparagine, aspartic acid,glutamic acid, glutamine, histidine, lysine, serine, threonine, andtheir homologs.

"Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic oraromatic amino acid, such as isoleucine, leucine, methionine,phenylalanine, tryptophan, tyrosine, valine, and their homologs.

For the purposes of this invention, alanine is classified as"amphiphilic" i.e., capable of acting as either hydrophilic orlipophilic. "Physiologically active truncated homolog or analog of PTHor PTHrp" refers to a polypeptide having a sequence comprising less thanthe full complement of amino acids found in PTH or PTHrp which, however,elicits a similar physiological response. The truncated PTH or PTHrpneed not be fully homologous with PTH or PTHrp to elicit a similarphysiological response. PTH(1-34) and PTHrp(1-34) are preferred, but notexclusive, representatives of this group. "Amphipathic α-helix" refersto the secondary structure exhibited by certain polypeptides in whichthe amino acids assume an α-helical configuration having opposing polarand nonpolar faces oriented along the long axis of the helix. Thepossibility of α-helical structure in the polypeptide of interest may beexplored to some extent by the construction of a "Schiffer-Edmundsonwheel" (M. Schiffer and A. B. Edmundson, Biophys. J. 7, 121 (1967)), ofthe appropriate pitch and noting the segregation of the hydrophilic andlipophilic residues on opposite faces of the cylinder circumscribing thehelix. Alternatively, empirical evidence, such as circular dichroism orx-ray diffraction data, may be available indicating the presence of anα-helical region in a given polypeptide. An ideal α-helix has 3.6 aminoacid residues per turn with adjacent side chains separated by 100° ofarc. Eisenberg et al. in Nature 299:371-374 (1982) and Proc. Nat. Acad.Sci. USA 81:140-144 (1984) have combined a hydrophobicity scale with thehelical wheel to quantify the concept of amphipathic helices. The meanhydrophobic moment is defined as the vector sum of the hydrophobicitiesof the component amino acids making up the helix. The followinghydrophobicities for the amino acids are those reported by Eisenberg(1984) as the "consensus" scale:

Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37;

Met 0.26 Ala 0.25; Gly 0.16; Cys 0.04; Tyr 0.02;

Pro -0.07; Thr -0.18; Ser -0.26; His -0.40; Glu -0.62;

Asn -0.64; Gln -0.69; Asp -0.72; Lys -1.10; Arg -1.76.

The hydrophobic moment, μ_(H), for an ideal α-helix having 3.6 residuesper turn (or a 100° arc (=360°/3.6) between side chains), may becalculated from:

    μ.sub.H = (ΣH.sub.N sin δ (N-1).sup.2 +(ΣH.sub.N cos δ (N-1)).sup.2 !.sup.1/2,

where H_(N) is the hydrophobicity value of the N^(th) amino acid and thesums are taken over the N amino acids in the sequence with periodicityδ=100°. The hydrophobic moment may be expressed as the mean hydrophobicmoment per residue by dividing μ_(H) by N to obtain <μ_(H) >. A value of<μ_(H) > at 100°±20° of about 0.20 or greater is suggestive ofamphipathic helix formation. The <μ_(H) > values at 100° for hPTHrp(22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.

Cornett, et al., in J. Mol. Biol., 195:659-685 (1987) have furtherextended the study of amphiphathic α-helices by introducing the"amphipathic index" as a predictor of amphipathicity. They concludedthat approximately half of all known α-helices are amphipathic, and thatthe dominant frequency is 97.5° rather than 100°, with the number ofresidues per turn being closer to 3.7 than 3.6. While such refinementsare scientifically interesting, the basic approach of Eisenberg, et al.is sufficient to classify a given sequence as amphipathic, particularlywhen one is designing a sequence ab initio to form an amphipathicα-helix.

A substitute amphipathic α-helical amino acid sequence may lack homologywith the sequence of a given segment of a naturally occurringpolypeptide but elicits a similar secondary structure, i.e. an α-helixhaving opposing polar and nonpolar faces, in the physiologicalenvironment. Replacement of the naturally occurring amino acid sequencewith an alternative sequence may beneficially affect the physiologicalactivity, stability, or other properties of the altered parentpolypeptide. Guidance as to the design and selection of such sequencesis provided in J. L. Krstenansky, et al., FEBS Letters 242:2, 409-413(1989), and J. P. Segrest, et al. Proteins: Structure, Function, andGenetics 8:103-117 (1990) among others.

The ten amino acid amphipathic α-helix of this invention has theformula:

    Haa (Laa Laa Haa Haa).sub.2 Laa

wherein the Haa's are selected from the group of hydrophilic amino acidsand the Laa's are selected from the group of lipophilic amino acids, asdefined above. Assuming an idealized α-helix, residues 1, 4, 5, 8, and 9are distributed along one face (A) of the helix within about a 140° arcof each other, while residues 2, 3, 6, 7, and 10 occupy an opposing 140°arc on the other face (B) of the helix. Preferably, all the residues onone face are of the same polarity while all those on the other face areof the opposite polarity, i.e., if face A is all hydrophilic, face B isall lipophilic and vice versa. The skilled artisan will recognize thatwhile the helices of this invention are described by

    Haa(Laa Laa Haa Haa) .sub.2 Laa,

the reverse sequence,

    Laa (Haa Haa Laa Laa).sub.2 Haa

will also meet the residue distribution criteria and is an equivalentdescriptor of the helices of this invention.

Alanine may be substituted for either hydrophilic or lipophilic aminoacids, since Ala can reside readily on either face of an amphipathicα-helix, although Ala₁₀ does not form an amphipathic α-helix. Generally,proline, cysteine, and tyrosine are not used; however, their presenceand other random errors in the sequence may be tolerated, e.g. ahydrophilic residue on the lipophilic face, as long as the remainingamino acids in the segment substantially conform to the hydrophilicface--lipophilic face division. A convenient method for determining if asequence is sufficiently amphipathic to be a sequence of this inventionis to calculate the mean hydrophobic moment, as defined above. If thepeak mean moment per residue at 100°±20° exceeds about 0.20, then thesequence will form an amphipathic helix and is a sequence of thisinvention.

For example, the mean hydrophobic moment per residue at 100° for (SEQ IDNO: 26), Xaa=Glu, is calculated as follows:

    ______________________________________                                        A.A.  H.sub.N  δ (N - 1)                                                                         H sin δ(N - 1)                                                                    H cos δ(N - 1)                       ______________________________________                                        E     -.62      0        0         -.62                                       L     .53      100       .52       -.17                                       L     .53      200       -.18      -.50                                       E     -.62     300       .34       -.31                                       K     -1.1     400       -.70      -.85                                       L     .53      500       .34       -.41                                       L     .53      600       -.46      -.27                                       E     -.62     700       .21       -.58                                       K     -1.1     800       -1.08     -.19                                       L     .53      900       0         -.53                                                                Σ = 0.81                                                                          Σ = -4.43                            μ.sub.H  =  (0.81).sup.2  + (-4.43).sup.2 !.sup.1/2  = 4.50                < μ.sub.H  > = 4.50/10 = 0.45                                              ______________________________________                                    

For this sequence, the mean peak hydrophobic moment occurs at 92° andhas a value of 0.48.

In applying this concept to parathyroid hormone and parathyroid hormonerelated peptide, it was hypothesized that either or both regions (7-16)and (22-31) may exhibit α-helical secondary structure and could bereplaced with a non-homologous sequence having similar structuraltendencies, without loss of biological activity or induction ofimmunoreaction.

Preferred Embodiments

In one aspect, this invention provides analogs of PTH, PTHrp, and thephysiologically active truncated analogs and homologs of PTH and PTHrp,or salts thereof, in which amino acid residues (22-31) form anamphipathic α-helix, the sequence of said residues (22-31) selectedfrom: ##STR11## wherein Xaa¹ and Xaa⁴ are independently Glu, Glu(OCH₃),His, or Phe; Xaa² is Leu or Phe; Xaa⁵ is Lys or His; Xaa⁷ and Xaa¹⁰ areindependently Leu or Ile; Xaa⁸ is Ala, Arg, or Glu; and Xaa⁹ is Lys orGlu (SEQ ID NO: 85); preferably ##STR12## wherein Xaa is Glu or Arg (SEQID NO:26); ##STR13## wherein Xaa¹ and Xaa⁴ are independently Glu,Glu(OCH₃), His, or Phe; Xaa² is Leu or Phe; Xaa⁸ is Glu, Lys, orLys(COCH₂ PEGX) and PEGX is a poly-(ethylene glycol methyl ether)radical of molecular weight 100 to 10,000 (SEQ ID NO:86); preferably,##STR14## wherein Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is apoly-(ethylene glycol methyl ether) radical of molecular weight 100 to10,000 (SEQ ID NO:27); ##STR15##

In another aspect, this invention provides analogs of PTH, PTHrp, andthe physiologically active truncated analogs and homologs of PTH andPTHrp, or salts thereof, of the formula:

Xaa¹ Xaa² Xaa³ Xaa⁴ Xaa⁵ Xaa⁶ Xaa⁷ Leu His Xaa¹⁰ Xaa¹¹ Gly Xaa¹³ Ser IleGln Xaa¹⁷ Leu Xaa¹⁹ Xaa²⁰ Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Xaa³⁵ Xaa³⁶Xaa³⁷ Xaa³⁸ Term, wherein:

Xaa¹ is absent or is Ala;

Xaa² is absent or is Val;

Xaa³ is absent or is Ser;

Xaa⁴ is absent or is Glu or Glu(OCH₃);

Xaa⁵ is absent or is His or Ala;

Xaa⁶ is absent or is Gln;

Xaa⁷ is absent or is Leu;

Xaa¹⁰ and Xaa¹⁷ are independently Asp or Asp(OCH₃);

Xaa¹¹ is Lys, Arg, or Leu;

Xaa¹³ is Lys, Arg, Tyr, Cys, Leu, Cys(CH₂ CONH(CH₂)₂ NH(biotinyl)),Lys(7-dimethylamino-2-oxo-2H-1-benxopyran-4-acetyl), orLys(dihydrocinnamoyl);

Xaa²⁰ is Arg or Leu;

Xaa¹⁹ and Xaa²¹ are independently Lys, Ala, or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID NOS:26, 27, 28, 29, or 30);

Xaa³² is His, Pro, or Lys;

Xaa³³ is absent, or is Pro, Thr, Glu, or Ala;

Xaa³⁴ is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, Leu,or 1,4-diaminobutyryl lactam;

Xaa³⁵ is absent or is Pro, Glu, Ser, Ala, or Gly;

Xaa³⁶ is absent or is Ala, Arg, or Ile;

Xaa³⁷ is absent or is Arg, Trp, or 3-(-2-naphthyl)-L-alanine;

Xaa³⁸ is absent or is Ala or hSer or Xaa³⁸⁻⁴² is Thr Arg Ser

and Term is OR or NR₂ where each R is independently H, (C₁ -C₄)alkyl orphenyl(C₁ -C₄)alkyl; and the pharmaceutically acceptable salts thereof.

In yet another aspect this invention includes polypeptide analogs of thephysiologically active truncated homolog hPTHrp(1-34), as shown inFormula (I):

Ala Val Ser Glu Xaa⁵ Gln Leu Leu His Asp Xaa¹¹ Gly Xaa¹³ Ser Ile Gln AspLeu Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Term,

wherein:

Xaa⁵ is His or Ala;

Xaa¹¹ and Xaa¹³ are independently Lys, Arg, or Leu; Xaa¹⁹ and Xaa²¹ areindependently Ala or Arg;

Xaa²²⁻³¹ is selected from: ##STR16## Xaa is Glu or Arg (SEQ ID NO:26);##STR17## wherein Xaa is Glu, Lys, or Lys(COCH₂ PEGX) and PEGX is apoly(ethylene glycol methyl ether) radical of molecular weight 100 to10,000 (SEQ ID NO:27); ##STR18## Xaa³² is His or Lys; Xaa³³ is Thr, Glu,or Ala;

Xaa³⁴ is Ala, hSer, Tyr, or Leu;

and Term is Gly Arg Arg, lactone, OH or NR₂, where each R is H or (C₁-C₄) alkyl; and their pharmaceutically acceptable salts. (Formula I)

A more specific aspect of the invention includes those polypeptides ofFormula (I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26), for which <μ_(H) > at100° exceeds 0.45. A still more specific aspect of the inventionincludes those Formula (I) polypeptides wherein Xaa²²⁻³¹ is (SEQ IDNO:26); Xaa¹¹ and Xaa¹³ are both Lys; and Xaa¹⁹ and Xaa²¹ are both Arg.

Representative polypeptides include, but are not limited to: ##STR19##

Another aspect of this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); Xaa¹¹ and Xaa¹³ are both Lys;and one of Xaa¹⁹ and Xaa²¹ is Arg and the other is Ala. Representativepolypeptides of this subgenus include, but are not limited to: ##STR20##

In another aspect this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO:26); one of Xaa¹¹ and Xaa¹³ is Leuand the other is Lys; and Xaa¹⁹ and Xaa²¹ are both Arg. Representativepolypeptides of this subgenus include, but are not limited to: ##STR21##

In another aspect this invention includes those polypeptides of Formula(I) wherein Xaa²²⁻³¹ is (SEQ ID NO:27), for which <μ_(H) > at 100°exceeds 0.50. A further aspect of this invention includes those Formula(I) polypeptides wherein Xaa²²⁻³¹ is (SEQ ID NO:27); Xaa¹¹ and Xaa¹³ areboth Lys or both Arg; and Xaa²¹ and Xaa²¹ are both Arg. Representativepolypeptides of this subgenus include, but are not limited to: ##STR22##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:28), for which <μ_(H) > at 100° is about0.25. Representative polypeptides of this subgenus include, but are notlimited to: ##STR23##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:29), for which <μ_(H) > at 100° is about0.28. Representative polypeptides of this subgenus include, but are notlimited to: ##STR24##

In another aspect this invention includes polypeptides of Formula (I)wherein Xaa²²⁻³¹ is (SEQ ID NO:30), for which <μ_(H) > at 100° is about0.29. Representative polypeptides of this subgenus include, but are notlimited to: ##STR25##

Still another aspect of this invention includes polypeptide analogs ofthe physiologically active truncated homolog bPTH(1-34), as shown inFormula (II):

Xaa¹ Val Ser Glu Ile Gln Xaa⁷ Xaa⁸ His Asn Leu Gly Lys His Leu Xaa¹⁶ SerXaa¹⁸ Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ His Asn Xaa³⁴ Term,

wherein:

Xaa¹ is Ser or Ala;

Xaa⁷ is Leu or Phe;

Xaa⁸ is Met or Nle;

Xaa¹⁶ is Asn or Ser;

Xaa¹⁸ is Leu, Met, or Nle;

Xaa¹⁹ is Glu or Arg;

Xaa²¹ is Val or Arg;

Xaa²²⁻³¹ is selected from (SEQ ID NO:26, 27, 28, 29, and 30);

Xaa³⁴ is Phe or Tyr;

Term is OH or NR₂, where each R is H or (C₁ -C₄)alkyl; and thepharmaceutically acceptable salts thereof. (Formula II) Representativepolypeptides include, but are not limited to: ##STR26##

In still another aspect of this invention, it has surprisingly beenfound that homologs and analogs of PTH and PTHrP having less than 34amino acids are also potent bone remodeling agents. These compounds areof general formula:

Ala Val Ser Glu Xaa⁵ Gln Leu Leu His Asp Xaa¹¹ Gly Xaa¹³ Ser Ile Gln AspLeu Xaa¹⁹ Arg Xaa²¹ Xaa²²⁻³¹ Xaa³² Xaa³³ Xaa³⁴ Term,

Representative polypeptides include, but are not limited to: ##STR27##Physical Data

m.p. 142.8°-166.1° C. α!_(D) ²⁵ -53.80 (c 0.38, H₂ O)

FAB (C₁₇₃ H₂₉₅ N₅₅ _(O) ₄₉): M+H!⁺ 3929

AAA: Asx 2.0 (2) Glx 5.7 (6) Ser 1.8 (2) His 3.0 (3) Gly 1.1 (1) Ala 0.9(1) Arg 2.8 (3) Val 1.2 (1) Ile 0.9 (1) Leu 7.4 (8) Lys 4.4 (4) Pro 0.9(1) ##STR28## Physical Data

m.p. 161.0°-177.0° C. α!_(D) ²⁵ -61.97 (c 0.19, H₂ O)

FAB (C₁₆₇ H₂₈₈ N₅₂ O₄₈): M+H!⁺ 3792.0

AAA: Asx 2.2 (2) Glx 5.9 (6) Ser 1.9 (2) His 2.1 (2) Gly 1.1 (1) Ala 1.0(1) Arg 3.0 (3) Val 1.1 (1) Ile 1.0 (1) Leu 7.9 (8) Lys 4.3 (4) Pro 0.9(1)

The skilled artisan will appreciate that numerous permutations of thepolypeptide analogs may be synthesized which will possess the desirableattributes of those described herein provided that an amino acidsequence having a mean hydrophobic moment per residue at 100°±20°greater than about 0.20 is inserted at positions (22-31).

Classical Synthesis of the Polypeptides

The polypeptides of the instant invention may be synthesized by methodssuch as those set forth in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984)and J. Meienhofer, Hormonal Proteins and Peptides, Vol. 2, AcademicPress, New York, (1973) for solid phase synthesis and E. Schroder and K.Lubke, The Peptides, Vol. 1, Academic Press, New York, (1965) forsolution synthesis.

In general, these methods involve the sequential addition of protectedamino acids to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid and any reactive side chain groupare protected. This protected amino acid is then either attached to aninert solid support, or utilized in solution, and the next amino acid inthe sequence, also suitably protected, is added under conditionsamenable to formation of the amide linkage. After all the desired aminoacids have been linked in the proper sequence, protecting groups and anysolid support are removed to afford the crude polypeptide. Thepolypeptide is desalted and purified, preferably chromatographically, toyield the final product.

A preferred method of preparing the analogs of the physiologicallyactive truncated polypeptides, having fewer than about forty aminoacids, involves solid phase peptide synthesis. In this method theα-amino (N.sup.α) functions and any reactive side chains are protectedby acid- or base-sensitive groups. The protecting group should be stableto the conditions of peptide linkage formation, while being readilyremovable without affecting the extant polypeptide chain. Suitableα-amino protecting groups include, but are not limited tot-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz),o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl,t-amyloxycarbonyl (Amoc), isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxy-carbonyl, o-nitrophenylsulfenyl,2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and thelike, preferably t-butoxycarbonyl (Boc). Suitable side chain protectinggroups include, but are not limited to: acetyl, benzyl (Bzl),benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl,t-butyldimethylsilyl, 2-chlorobenzyl (Cl-z), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl(Tos), trimethylsilyl, and trityl.

In solid phase synthesis, the C-terminal amino acid is first attached toa suitable resin support. Suitable resin supports are those materialswhich are inert to the reagents and reaction conditions of the stepwisecondensation and deprotection reactions, as well as being insoluble inthe media used. Examples of commercially available resins includestyrene/divinylbenzene resins modified with a reactive group, e.g.,chloromethylated co-poly-(styrene-divinyl-benzene), hydroxymethylatedco-poly-(styrene-divinyl-benzene), and the like. Benzylated,hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred. Whenthe C-terminus of the compound is an amide, a preferred resin isp-methylbenzhydrylamino-co-poly(styrene-divinyl-benzene) resin.

Attachment to the PAM resin may be accomplished by reaction of theN.sup.α -protected amino acid, preferably the Boc-amino acid, as itsammonium, cesium, triethylammonium, 1,5-diazabicyclo- 5.4.0!undec-5-ene,tetramethylammonium, or similar salt in ethanol, acetonitrile,N,N-dimethylformamide (DMF), and the like, preferably the cesium salt inDMF, with the resin at an elevated temperature, for example betweenabout 40° and 60° C., preferably about 50° C., for from about 12 to 72hours, preferably about 48 hours.

The N.sup.α -Boc-amino acid may be attached to the benzhydrylamine resinby means of, for example, an N,N'-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2to about 24 hours, preferably about 2 hours at a temperature of betweenabout 10° and 50° C., preferably 25° C. in a solvent such asdichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids may be carried out bymethods well known in the art, typically in an automated peptidesynthesizer. Following neutralization with triethylamine or similarbase, each protected amino acid is preferably introduced inapproximately 1.5 to 2.5 fold molar excess and the coupling carried outin an inert, nonaqueous, polar solvent such as dichloromethane, DMF, ormixtures thereof, preferably in dichloromethane at ambient temperature.Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DIC),N,N'-diisopropylcarbodiimide (DIC) or other carbodiimide, either aloneor in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl ureas,benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBop), N-hydroxysuccinimide, other N-hydroxyimides, or oximes.Alternatively, protected amino acid active esters (e.g. p-nitrophenyl,pentafluorophenyl and the like) or symmetrical anhydrides may be used.

At the end of the solid phase synthesis the fully protected peptide isremoved from the resin. When the linkage to the resin support is of thebenzyl ester type, cleavage may be effected by means of aminolysis withan alkylamine or fluoroalkylamine for peptides with an alkylamideC-terminus, or by aminolysis with, for example, ammonia/methanol orammonia/ethanol for peptides with an unsubstituted amide C-terminus, ata temperature between about -10° and 50° C., preferably about 25° C.,for between about 12 and 24 hours, preferably about 18 hours. Peptideswith a hydroxy C-terminus may be cleaved by HF or other strongly acidicdeprotection regimen or by saponification. is Alternatively, the peptidemay be removed from the resin by transesterification, e.g., withmethanol, followed by aminolysis or saponification. The protectedpeptide may be purified by silica gel chromatography.

The side chain protecting groups may be removed from the peptide bytreating the aminolysis product with, for example, anhydrous liquidhydrogen fluoride in the presence of anisole or other carbonium ionscavenger, treatment with hydrogen fluoride/pyridine complex, treatmentwith tris(trifluoroacetyl)boron and trifluoroacetic acid, by reductionwith hydrogen and palladium on carbon or polyvinylpyrrolidone, or byreduction with sodium in liquid ammonia, preferably with liquid hydrogenfluoride and anisole at a temperature between about -10° and +10° C.,preferably at about 0° C., for between about 15 minutes and 2 hours,preferably about 1.5 hours. For peptides on the benzhydrylamine resin,the resin cleavage and deprotection steps may be combined in a singlestep utilizing liquid hydrogen fluoride and anisole as described above.

The solution may be desalted (e.g. with BioRad AG-3® anion exchangeresin) and the peptide purified by a sequence of chromatographic stepsemploying any or all of the following types: ion exchange on a weaklybasic resin in the acetate form; hydrophobic adsorption chromatographyon underivatized co-poly(styrene-divinylbenzene), e.g. Amberlite® XAD;silica gel adsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g. on Sephadex®G-25; countercurrent distribution; or high performance liquidchromatography (HPLC), especially reversed-phase HPLC on octyl- oroctadecylsilylsilica (ODS) bonded phase column packing.

Thus, another aspect of the present invention relates to processes forpreparing polypeptides and pharmaceutically acceptable salts thereofwhich processes comprise sequentially condensing protected amino acidson a suitable resin support, removing the protecting groups and resinsupport, and purifying the product, to afford analogs of thephysiologically active truncated homologs and analogs of PTH and PTHrp,preferably of PTH(1-34) and PTHrp(1-34), in which the amino acids atpositions (22-31) form an amphipathic α-helical peptide sequence, asdefined above.

Recombinant Synthesis of the Polypeptides

Alternatively, the polypeptides of this invention may be prepared bycloning and expression of a gene encoding for the desired polypeptide.In this process, a plasmid containing the desired DNA sequence isprepared and inserted into an appropriate host microorganism, typicallya bacteria, such as E. coli, or a yeast, such as Saccharomycescerevisiae, inducing the host microorganism to produce multiple copiesof the plasmid, and so of the cDNA encoding for the polypeptide analogsof this invention.

First, a synthetic gene coding for the selected PTH or PTHrp analog isdesigned with convenient restriction enzyme cleavage sites to facilitatesubsequent alterations. Polymerase chain reaction (PCR), as taught byMullis in U.S. Pat. Nos. 4,683,195 and 4,683,202, may be used to amplifythe sequence.

The amplified synthetic gene may be isolated and ligated to a suitableplasmid, such as a Trp LE plasmid, into which four copies of the genemay be inserted in tandem. Preparation of Trp LE plasmids is describedin U.S. Pat. No. 4,738,921 and European Pat. Publication No. 0212532.Trp LE plasmids generally produce 8-10 times more protein than Trp Eplasmids. The multi-copy gene may then be expressed in an appropriatehost, such as E. coli or S. cerevisiae.

The specific expression vector used herein was Trp LE 18 Prot (Ile³,Pro⁵) containing the following elements: a pBR322 fragment (EcoRI-BamHI)containing the ampicillin resistant gene and the plasmid origin ofreplication; an EcoRI-SacII fragment containing the trp promoter and thetrpE gene; an HIV protease (Ile3,Pro⁵) gene fragment (SacII-HindIII); abGRF gene fragment (HindIII-BamHI); and a transcription terminator fromE. coli rpoc gene. The HIV protease and bGRF gene fragments are notcritical and may be replaced with other coding sequences, if desired.

The expressed multimeric fusion proteins accumulate intracellularly intostable inclusion bodies and may be separated by centrifugation from therest of the cellular protein. The isolated fusion protein is convertedto the monomeric PTH or PTHrp analog and may be purified by cationexchange and/or reverse phase HPLC.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Maniatis, et al., Molecular Cloning, aLaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).

Utility and Administration

The polypeptides of this invention are useful for the prevention andtreatment of a variety of mammalian conditions manifested by loss ofbone mass. In particular, the compounds of this invention are indicatedfor the prophylaxis and therapeutic treatment of osteoporosis andosteopenia in humans.

In general, the polypeptides of this invention, or salts thereof, areadministered in amounts between about 0.002 and 1 μg/kg body weight perday, preferably from about 0.04 to about 0.2 μg/kg body weight per day.For a 50 kg human female subject, the daily dose of active ingredient isfrom about 0.1 to about 50 μgs, preferably from about 2.0 to about 10μgs. In other mammals, such as horses, dogs, and cattle, higher dosesmay be required. This dosage may be delivered in a conventionalpharmaceutical composition by a single administration, by multipleapplications, or via controlled release, as needed to achieve the mosteffective results, preferably one or more times daily by injection.

The selection of the exact dose and composition and the most appropriatedelivery regimen will be influenced by, inter alia, the pharmacologicalproperties of the selected polypeptide, the nature and severity of thecondition being treated, and the physical condition and mental acuity ofthe recipient.

Representative delivery regimens include oral, parenteral (includingsubcutaneous, intramuscular and intravenous), rectal, buccal (includingsublingual), pulmonary, transdermal, and intranasal.

Pharmaceutically acceptable salts retain the desired biological activityof the parent polypeptide without toxic side effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like; and salts formed with organic acids suchas, for example, acetic acid, oxalic acid, tartaric acid, succinic acid,maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonicacids, polygalacturonic acid and the like; (b) base addition saltsformed with polyvalent metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and thelike; or with an organic cation formed from N,N'-dibenzylethylenediamineor ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinctannate salt and the like.

A further aspect of the present invention relates to pharmaceuticalcompositions comprising as an active ingredient a polypeptide of thepresent invention, or pharmaceutically acceptable salt thereof, inadmixture with a pharmaceutically acceptable, non-toxic carrier. Asmentioned above, such compositions may be prepared for parenteral(subcutaneous, intramuscular or intravenous) administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for pulmonary or intranasal administration, particularly in the form ofpowders, nasal drops or aerosols; and for rectal or transdermaladministration.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985). Formulations for parenteral administration may contain asexcipients sterile water or saline, alkylene glycols such as propyleneglycol, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes and the like. For oraladministration, the formulation can be enhanced by the addition of bilesalts or acylcarnitines. Formulations for nasal administration may besolid and may contain excipients, for example, lactose or dextran, ormay be aqueous or oily solutions for use in the form of nasal drops ormetered spray. For buccal administration typical excipients includesugars, calcium stearate, magnesium stearate, pregelatinated starch, andthe like.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be enhanced by surfactant acids, such as forexample, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,glycodeoxycholic acid, cyclodextrins and the like in an amount in therange between about 0.2 and 15 weight percent, preferably between about0.5 and 4 weight percent, most preferably about 2 weight percent.

Delivery of the compounds of the present invention to the subject overprolonged periods of time, for example, for periods of one week to oneyear, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, osmotic pumps,vesicles, micelles, liposomes, transdermal patches, iontophoreticdevices and alternative injectable dosage forms may be utilized for thispurpose. Localization at the site to which delivery of the activeingredient is desired is an additional feature of some controlledrelease devices, which may prove beneficial in the treatment of certaindisorders.

One form of controlled release formulation contains the polypeptide orits salt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. Pat. No.4,675,189. The compounds or, preferably, their relatively insolublesalts, may also be formulated in cholesterol or other lipid matrixpellets, or silastomer matrix implants. Additional slow release, depotimplant or injectable formulations will be apparent to the skilledartisan. See, for example, Sustained and Controlled Release DrugDelivery Systems, J. R. Robinson ed., Marcel Dekker, Inc., New York,1978, and R. W. Baker, Controlled Release of Biologically Active Agents,John Wiley & Sons, New York, 1987.

The following specific Examples are intended to illustrate the synthesisand testing of representative compounds of the invention and should notbe construed as limiting the scope of the claims. In the Examples,"m.p." is melting point, " α!_(D) ²⁵ " is the optical activity at 25° C.at the given concentration in the indicated solvent, "FAB" is fast atombombardment mass spectrometry, and "AAA" is amino acid analysis, withexpected values in parentheses following the observed values. The aminoacid analyses were conducted on a Hewlett-Packard AminoQuant Analyzerfollowing the manufacturer's recommended protocols. Primary amino acidswere derivatized with o-phthalaldehyde; secondary amino acids with Fmoc.Fluorescent detection of the derivatized amino acids was used forquantification. The protected amino acids were obtained from AppliedBiosystems Inc. (Foster City, Calif.).

EXAMPLE I

Compound 1 (SEQ ID NO:7) was prepared on 0.5 mmol scale by the solidphase method on 4-methylbenzhydrylamine resin, using an automatedApplied Biosystems Model 430A peptide synthesizer. The α-amino groupswere protected with t-butoxycarbonyl (Boc). The side chain protectinggroups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Tos) for Arg;benzyloxymethyl (Bom) for His; and 2-chlorobenzyl (Cl-z) for Lys. Theamino acids were coupled sequentially usingN,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) followingStewart and Young (supra). After each amino acid coupling, the peptidewas acetylated using a mixture of acetic anhydride anddiisopropylethylamine in N-methylpyrrolidone. The completed peptide wascleaved from the resin with simultaneous deprotection of the side chain,protecting groups using anhydrous HF (25 mL) in the presence of anisole(2.5 mL) at -10° C. for 30 minutes and at 0° C. for 60 minutes. Afterevaporation of the HF in vacuo, the is residue was washed with anhydrousether, and the crude peptide extracted with 10% acetic acid.Lyophilization of the 10% acetic acid extract gave 900 mgs of crudeproduct. The peptide was purified by medium pressure ODS reversed phasecolumn chromatography using a gradient of 22-45% CH₃ CN in 0.1% TFA. Theproduct eluted in three fractions, which were concentrated andlyophilized to give 130 mgs of white solid of >98% purity. ##STR29##Physical Data

m.p. 150°-159° C. α!_(D) ²⁵ -34.88° (c 0.16, H₂ O)

FAB (C₁₇₅ H₃₀₀ N₅₆ O₅₁): M+H!⁺ 4005.5

AAA: Asp, 1.9(2); Glu, 5.6(6); Ser, 1.6(2); His, 2.7(3); Gly, 1.0(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.0(1); Ile, 0.9(1); Leu,7.3(8); Lys, 4.0(4).

Similarly, Compounds 2, 5-18, 21-27, 29-36, 38-48, 50-54, 58-64 and66-70 were prepared and characterized, substituting PAM resin as neededfor the synthesis of hydroxy-terminal polypeptides. ##STR30## PhysicalData

m.p. 154°-170° C. α!_(D) ²⁵ -49.35° (c 0.46, H₂ O)

FAB (C₁₇₅ H₃₀₁ N₅₇ O₅₀): M+H!⁺ 4005.0

AAA: Asp, 2.1(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly 1.1(1);Thr, 1.0(1); Ala, 1.9(2); Arg, 3.0(3); Val, 1.2(1); Ile, 1.0(1); Leu,7.8(8); Lys, 4.2(4). ##STR31## Physical Data

m.p. 147°-165° C. α!_(D) ²⁵ -49.17° (c 0.66, H₂ O)

FAB (C₁₇₅ H₂₉₉ N₅₉ O₅₂): M+H!⁺ 4061

AAA: Asp, 2.1(2); Gly, 6.1(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5); Val, 1.0(1); Ile, 0.9(1); Leu,7.7(8); Lys, 1.9(2). ##STR32## Physical Data

m.p. 150°-170° C. α!_(D) ²⁵ -48.65° (c. 0.54, H₂ O)

FAB (C₁₇₅ H₂₉₉ N₆₃ O₅₂): M+H!⁺ 4118.0

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.2(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 6.9(7); Val, 1.0(1); Ile, 1.0(1); Leu,7.8(8). ##STR33## Physical Data

m.p. 177°-182° C. α!_(D) ²⁵ -46.17° (c 0.14, H₂ O)

FAB (C₁₇₆ H₃₀₄ N₆₄ O₅₀): M+H!⁺ 4117

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7(7); Val, 1.0(1); Ile, 1.0(1); Lys,7.7(8); Lys, 0.9(1). ##STR34## Physical Data

m.p. 147°-165° C. α!_(D) ²⁵ - 49.17° (c 0.66, H₂ O)

FAB (C₁₇₆ H₃₀₅ N₅₉ O₄₉): M+H!⁺ 4033.0

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 2.7(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4); Val, 1.0(1); Ile, 1.0(1); Leu,7.9(8); Lys, 4.0(4). ##STR35## Physical Data

m.p. 158°-160° C. α!_(D) ²⁵ -44.76° (c 0.1, H₂ O)

FAB (C₁₈₉ H₃₂₆ N₆₄ O₅₅): M!⁺ 4375.0

AAA: Asp, 2.0(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 2.3(2);Thr, 1.0(1); Ala, 1.9(2); Arg, 5.0(5); Val, 1.2(1); Ile, 1.0(1); Leu,7.8(8); Lys, 4.3(4). ##STR36## Physical Data

m.p. 170°-175° C. α!_(D) ²⁵ -31.59° (c 0.54, H₂ O)

FAB (C₁₇₄ H₃₀₀ N₅₂ O₅₁): M+!₊ 3936.0

AAA: Asp, 2.0(2); Glu, 6.0(6); Ser, 1.8(2); His, 2.0(2); Gly, 1.2(1);Ala, 3.0(3); Arg, 2.8(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.9(10); Lys,3.0(3). ##STR37## Physical Data

m.p. 172°-174° C. α!_(D) ²⁵ -43.29° (c 0.2, H₂ O)

FAB (C₁₇₉ H₃₁₁ N₅₅ O₅₂): !M+H!⁺ 4065.8

AAA: Asp, 2.2(2); Glu, 7.7(7); Ser, 1.7(2); His, 2.0(2); Gly, 1.0(1);Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.3(9); Lys,5.1(5). ##STR38## Physical Data

m.p. 178° C. α!_(D) ²⁵ -36.88° (c 0.4, H₂ O)

FAB (C₁₈₂ H₂₉₅ N₅₀ O₅₁): M+H!⁺ 4001.6

AAA: Asp, 2.1(2); Glu, 6.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.1(1);Ala, 1.0(1); Arg, 1.0(1); Tyr, 0.8(1); Val, 2.0(2); Phe, 1.0(1); Ile,0.9(1); Leu+Nle, 8.5(7+2); Lys, 3.1(3). ##STR39## Physical Data

m.p. 260° C. α!_(D) ²⁵ -37.02° (c 0.2, H₂ O)

FAB (C₁₈₄ H₃₀₄ N₅₆ O₄₉): M+H!⁺ 4084

AAA: Asp, 2.1(2); Glu, 5.5(5); Ser, 2.6(3); His, 3.1(3); Ala, 1.0(1);Gly, 1.1(1); Arg, 3.2(3); Tyr, 1.0(1); Val, 1.0(1); Phe, 1.0(1); Ile,1.0(1); Leu, 9.0(9); Lys, 3.0(3). ##STR40## Physical Data

m.p. 190°-225° C. α!_(D) ²⁵ -56.58° (c 0.36, H₂ O)

FAB (C161H₂₇₂ N₅₄ ₄₉): M+H!⁺ 3747.0

AAA: Asp, 2.1(2); Glu, 4.9(5); Ser, 1.7(2); His, 2.6(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3); Val, 1.2(1); Ile, 1.0(1); Leu,6.6(6); Lys, 1.9(2). ##STR41## Physical Data

m.p. 170°-180° C. α!_(D) ²⁵ -48.19° (c 0.2, H₂ O)

FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): M+H!⁺ 3919.0

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu,8.0(8); Lys, 4.4(4). ##STR42## Physical Data

m.p. 190°-195° C. α!_(D) ²⁵ -50.50° (c 0.4, H₂ O)

FAB (C₁₇₂ H₂₉₃ N₅₃ O₅₁): M+H!⁺ 3919.0

AAA: Asp, 2.1(2); Glu, 6.0(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu,7.5(8); Lys, 4.2(4). ##STR43## Physical Data

m.p. 195°-204° C. α!_(D) ²⁵ -67.11° (c 0.3, H₂ O)

FAB (C₁₆₃ H₂₈₀ N₅₄ O₅₀): M+H!⁺ 3796.0

AAA: Asp, 2.1(2); Glu, 2.9(3); Ser, 6.8(7); His, 3.1(3); Gly, 1.2(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.0(1); Ile, 1.0(1); Leu,8.2(8); Lys, 2.0(2). ##STR44## Physical Data

m.p. 200°-207° C. α!_(D) ²⁵ -60.26° (C 0.6, H₂ O)

FAB (C₁₇₄ H₂₈₄ N₅₆ O₅₅): M+H!+3960.0

AAA: Asp, 2.9(3), Glu, 3.5(4); Ser, 1.4(2); His, 2.6(3); Gly, 0.9(1);Thr, 1.0(1); Ala, 4.0(4); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.9(2); Phe,1.1(1); Ile, 0.9(1); Leu, 3.6(4); Lys, 4.1(4). ##STR45## Physical Data

m.p. 148-155° C. α!_(D) ²⁵ -45.97 (c 0.26, H₂ O)

FAB(C₁₈₄ H₃₀₄ N₅₆ O₄₉): M+H!⁺ 4084

AAA: Asx, 2.1(2); Glx, 5.0(5); Ser, 2.7(3); His, 3.0(3); Gly, 1.0(1);Ala, 0.9(1); Arg, 3.1(3); Tyr, 0.9(1); Val, 1.0(1); Phe, 0.9(1); Ile,0.9(1); Leu 9.3(9); Lys, 3.2(3). ##STR46## Physical Data

m.p. 175°-182° C. α!_(D) ²⁵ -49.99(c 0.47, H₂ O)

FAB(C₁₇₅ H₂₉₉ N₄₉ O₅₁): M+H!⁺ 3906.5

AAA: Asx, 2.1(2); Glx, 6.5(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 9.1(9); Lys,6.5(6). ##STR47## Physical Data

m.p. 136.5°-153.5° C. α_(D) ²⁵ -32.57(c 0.13, H₂ O)

FAB(C₁₇₅ H₃₀₀ N₆₀ O₅₁): M+H!⁺ 4060.8

AAA: Asx, 2.2(2); Glx, 6.2(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.1(2); Arg, 5.2(5); Val, 1.1(1); Ile, 1.1(1); Leu,8.4(8); Lys, 2.2(2). ##STR48## Physical Data

m.p. 125.8°-127.2° C. α!_(D) ²⁵ -54.62(c 0.23, H₂ O)

FAB(C₁₈₀ H₃₀₆ N₆₀ O₅₃): M+H!⁺ 4158.0

AAA: Asx, 2.1(2); Glx, 6.2(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 5.1(5); Val, 1.0(1); Ile, 1.0(1); Leu,8.0(8); Lys, 2.1(2); Pro, 1.1(1). ##STR49## Physical Data

m.p. 106°-137.3° C. α!_(D) ²⁵ -39.55(c 0.67, H₂ O)

FAB (C₁₈₉ H₃₂₆ N₆₈ O₅₅): M+H!⁺ 4430.5

AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 1.6(2); His, 2.7(3); Gly, 2.2(2);Thr, 1.0(1); Ala, 1.8(2); Arg, 7.3(7); Val, 0.8(1); Ile, 1.0(1); Leu,8.1(8); Lys, 2.1(2). ##STR50## Physical Data

m.p. 160°-172° C. α!_(D) ²⁵ -49.85(c 0.34, H₂ O)

FAB(C₁₈₁ H₃₀₄ N₅₆ O₅₂): M+H!⁺ 4096.9

AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 0.9(1); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.0(1); Ile,1.0(1); Leu, 7.7(8); Lys, 4.4(4). ##STR51## Physical Data

m.p. 130°-171° C. α!_(D) ²⁵ -40.65 (c 0.34, H₂ O)

FAB(C₁₇₈ H₂₉₇ N₅₅ O₅₂): M+!⁺ 4039.4

AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 3.4(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 2.9(3); Tyr, 0.8(1); Val, 1.0(1); Ile,0.9(1); Leu, 7.9(8); Lys, 3.4(3). ##STR52## Physical Data

m.p. 140°-160° C. α!_(D) ²⁵ -44.48 (c 0.25, H₂ O)

FAB(C₁₇₂ H₂₉₃ N₅₅ O₅₁ S₁): M+H!⁺ 3979

AAA: Asx+Cys, 3.0(2+1); Glx, 5.6(6); Ser, 1.7(2); His, 3.0(3); Gly,1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.5(3); Val, 1.0(1); Ile, 0.9(1);Leu, 7.5(8); Lys, 3.3(3). ##STR53## Physical Data

m.p. (not determined) α!_(D) ²⁵ (not determined)

FAB(C₁₈₆ H₃₁₆ N₅₉ O₅₄ S₂): M+H!⁺ 4306.6

AAA: Asx, 2.2(2); Glx, 6.1(6); Ser, 1.8(2); His, 3.8(3); Gly, 1.0(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu,8.3(3); Lys, 3.3(3). ##STR54## Physical Data

m.p. 135°-205° C. α! α!_(D) ²⁵ -26.92 (c 0.104, 50% aq. HOAC)

FAB(C₁₈₈ H₃₁₁ N₅₇ O₅₄): M+H!⁺ 4233

AAA: Asx, 1.9(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.2(3); Gly, 1.0(1);Thr, 1.1(1); Ala, 2.0(2); Arg, 3.2(3); Val, 1.1(1); Ile, 0.9(1); Leu,8.2(8); Lys, 4.5(4). ##STR55## Physical Data

m.p. 92.1°-146.6° C. α!_(D) ²⁵ -40.76 (c 0.34, H₂ O)

FAB(C₁₇₇ H₃₀₂ N₅₆ O₅₃): M+H!⁺ 4062.0

AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 2.2(2);Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.2(1); Ile, 0.9(1); Leu,7.5(8); Lys, 4.2(4). ##STR56## Physical Data

m.p. (not determined) α!_(D) ²⁵ -21.96 (c 0.132, H₂ O)

FAB(C₁₈₁ H₃₁₁ N₅₅ O₅₂): M+H!⁺ 4089.0

AAA: Asx, 2.1(2); Glx, 6.3(6); Ser, 1.8(2); His, 3.3(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu,8.0(8); Lys, 4.2(4). ##STR57## Physical Data

m.p. (not determined) α!_(D) ²⁵ -46.80 (c 0.07, H₂ O)

FAB (C₁₈₂ H₃₁₃ N₅₅ O₅₂): M+H!⁺ 4103

AAA: Asx, 2.1(2); Glx, 6.2(6); Ser, 1.4(2); His, 3.0(3); Gly, 1.1(1);Thr, 1.1(1); Ala, 1.7(2); Arg, 3.2(3); Val, 0.6(1); Ile, 0.9(1); Leu,8.0(8); Lys, 4.1(4). ##STR58## Physical Data

m.p. 152.1°-186.5° C. α!_(D) ²⁵ -55.91 (c 0.33, H₂ O)

FAB(C₁₈₀ H₃₀₈ N₅₆ O₅₃): M+H!⁺ 4102.6

AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 2.9(3); Val, 1.2(1); Ile, 1.0(1); Leu,7.7(8); Lys, 4.3(4). ##STR59## Physical Data

m.p. 120°-148.2° C. α!_(D) ²⁵ -52.78 (c 0.47, H₂ O)

FAB (C₁₇₇ H₃₀₁ N₅₅ O₅₂): M+H!⁺ 4031.0

AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.0(1);Thr, 1.0(1); Ala, 0.9(1); Arg, 2.9(3); Val, 1.2(1); Ile, 0.9(1); Leu,7.5(8); Lys, 3.6(3); Pro, 0.9(1). ##STR60## Physical Data

m.p. 133.9°-155.1° C. α!_(D) ²⁵ -54.22 (c 0.37, H₂ O)

FAB(C₁₇₇ H₃₀₂ N₅₆ O₅₁): M+H!⁺ 4030.7

AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 1.9(2); His, 2.9(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 0.9(1); Arg, 2.8(3); Val, 1.2(1); Ile, 1.1(1); Leu,7.8(8); Lys, 4.2(4); Pro, 0.9(1). ##STR61## Physical Data

m.p. 142.8°-166.1° C. α!_(D) ²⁵ -53.80 (c 0.38, H₂ O)

FAB (C₁₇₃ H₂₉₅ N₅₅ O₄₉): M+H!⁺ 3929

AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 1.1(1);Ala, 0.9(1); Arg, 2.8(3); Val, 1.2(1); Ile, 0.9(1); Leu, 7.4(8); Lys,4.4(4); Pro, 0.9(1). ##STR62## Physical Data

m.p. 161.0°-177.0° C. α!_(D) ²⁵ -61.97 (c 0.19, H₂ O)

FAB (C₁₆₇ H₂₈₈ N₅₂ O₄₈): M+H!⁺ 3792.0

AAA: Asx, 2.2(2); Glx, 5.9(6); Ser, 1.9(2); His, 2.1(2); Gly, 1.1(1);Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 7.9(8); Lys,4.3(4); Pro, 0.9(1). ##STR63## Physical Data

m.p. 181°-202° C. α!_(D) ²⁵ -45.14 (c 0.19, H₂ O)

FAB (C₁₉₅ H₃₂₆ N₆₂ O₅₆): M+H!⁺ 4435.2

AAA: Asx, 2.0(2); Glx, 5.8(6); Ser, 2.8(3); His, 2.8(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 3.7(4); Ile, 0.9(1); Leu, 7.5(8); Lys,4.3(4); Trp, 0.9(1). ##STR64## Physical Data

m.p. 130°-132.2° C. α!_(D) ²⁵ -46.66 (c 0.195, H₂ O)

FAB(C₂₁₆ H₃₆₅ N₈₁ O₆₂): M+H!⁺ 5088.8

AAA: Asx, 2.2(2); Glx, 6.0(6); Ser, 2.7(3); His, 3.0(3); Gly, 2.2(2);Thr, 2.1(2); Ala, 3.0(3); Arg, 10.5(10); Val, 0.9(1); Ile, 1.0(1); Leu,8.2(8); Trp, 1.0(1). ##STR65## Physical Data

m.p. 158°-174° C. α!_(D) ²⁵ -43.57 (c 0.53, H₂ O)

FAB (C₂₁₆ H₃₆₆ N₈₂ O₆₁): M+H!⁺ 5087.4

AAA: Asx, 1.9(2); Glx, 5.6(6); Ser, 2.6(3); His, 3.3(3); Gly, 2.1(2);Thr, 2.0(2); Ala, 2.9(3); Arg, 10.1(10); Val, 0.9(1); Ile, 1.0(1); Leu,8.3(8); Trp 1.1(1). ##STR66## Physical Data

m.p. 165.4°-175.2° C. α!_(D) ²⁵ -40.43 (c 0.20, H₂ O)

FAB (C₂₂₅ H₃₇₄ N₈₀ O₆₂): M+H!⁺ 5191

AAA: Asx, 2.1(2), Glx, 6.3(6); Ser, 2.8(3); His, 3.2(3); Gly, 2.1(2),Thr, 2.0(2); Ala, 3.2(3); Arg, 9.9(9); Val, 1.0(1), Ile, 0.9(1); Leu,8.6(8); Lys, 1.1(1); Trp, 1.1(1). ##STR67## Physical Data

m.p. 140°-160° C. α!_(D) ²⁵ -56.88 (c 0.16, H₂ O)

FAB(C₁₈₀ H₂₈₇ N₅₅ O₅₈): M+H!⁺ 3989.8

AAA: Asx, 3.0(3); Glx, 2.9(3); Ser, 3.7(4); His, 2.8(3); Gly, 1.1(1);Thr, 0.9(1); Ala, 1.9(2); Arg, 2.0(2); Tyr, 1.0(1); Val, 1.7(2); Phe,0.9(1); Ile, 0.9(1); Leu+Nle 5.8(2+4); Lys, 3.4(3); Trp, 1.1(1).##STR68## Physical Data

m.p. 140°-210° C. α!_(D) ²⁵ -47.75(c 0.178, H₂ O)

FAB(C₁₈₀ H₃₀₉ N₅₇ O₅₂ S₁): M+H!⁺ 4135.0

AAA: Asx, 2.3(2); Glx, 6.6(6); Ser, 1.4(2); His, 3.2(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 0.9(1); Met, 1.1(1); Ile,1.0(1); Leu, 8.8(8); Lys, 4.4(4). ##STR69## Physical Data

m.p. 136.5°-156.8° C. α!_(D) ²⁵ -49.89 (c 0.24, H₂ O)

FAB(C₁₈₂ H₃₀₀ N₅₆ O₄₉): M+H!⁺ 4056.0

AAA: Asx, 2.2(2); Glx, 5.0(5); Ser, 1.9(2); His, 3.3(3); Gly, 1.0(1);Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3); Val, 1.0(1); Phe, 2.0(2); Ile,0.9(1); Leu, 7.2(7); Lys, 3.5(4). ##STR70## Physical Data

m.p. 80.7°-141.0° C. α!_(D) ²⁵ -55.38 (c 0.23, H₂ O)

FAB (C₁₇₆ H₃₀₀ N₅₈ O₄₉): M+H!⁺ 4012.8

AAA: Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 4.3(4); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.0(1); Leu,8.1(8); Lys, 3.9(4). ##STR71## Physical Data

m.p. 134.3°-157.9° C. α!_(D) ²⁵ -50.72 (c 0.45, H₂ O)

FAB(C₁₇₅ H₂₉₅ N₅₇ O₅₁): M+H!⁺ 4012.8

AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 1.8(2); His, 4.2(4); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.1(1); Ile, 0.9(1); Leu,8.1(8); Lys, 3.1(3). ##STR72## Physical Data

m.p. 142.7°-159.8° C. α!_(D) ²⁵ -54.01 (c 0.21, H₂ O)

FAB(C₁₇₃ H₂₉₈ N₅₆ O₄₉) M+H!⁺ 3946.0

AAA: Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 3.1(3); Arg, 3.1(3); Val, 1.0(1); Ile, 1.9(2); Leu,7.0(7); Lys, 4.3(4). ##STR73## Physical Data

m.p. 138°-185° C. α!_(D) ²⁵ -50.17 (c 0.14, H₂ O)

FAB(C₁₇₄ H₂₉₅ N₅₅ O₅₃): M+H!⁺ 4005

AAA: Asx, 2.2(2); Glx, 7.1(7); Ser, 1.7(2); His, 2.8(3); Gly, 1.0(1);Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.7(2); Leu,7.1(7); Lys, 2.7(3). ##STR74## Physical Data

m.p. 158°-163° C. α!_(D) ²⁵ -46.06 (c 0.17, H₂ O)

FAB (C₁₉₅ H₃₂₇ N₆₃ O₅₅): M+H!⁺ 4434.8

AAA: Asx, 2.0(2); Glx, 5.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.0(1);Ala, 1.8(2); Arg, 4.0(4); Thr, 0.9(1); Val, 0.9(1); Ile, 0.9(1); Leu,7.5(8); Lys, 3.9(4); Trp, 1.0(1). ##STR75## Physical Data

m.p. 156°-162° C. α!_(D) ²⁵ -44.44 (c 0.189, H₂ O)

FAB (C₁₉₇ H₃₂₈ N₆₂ O₅₅): M+H!⁺ 4445.6

AAA: Asx, 2.1(2); Glx, 5.5(6); Ser, 2.8(3); His, 2.9(3); Gly, 1.0(1);Ala, 2.0(2); Arg, 4.0(4); Thr, 0.9(1); Val, 1.0(1); Ile, 0.9(1); Leu,7.5(8); Lys, 4.2(4); Nal, 1.1(1). ##STR76## Physical Data

m.p. 159°-164° C. α!_(D) ²⁵ -50.94 (c 0.29, H₂ O)

FAB (C₁₉₂ H₃₂₀ N₆₀ O₅₅): M+H!⁺ 4349.0

AAA: Asx, 2.0(2); Glx, 5.6(6); Ser, 2.7(3); His, 3.2(3); Gly, 1.0(1);Ala, 3.1(3); Arg, 2.8(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu,7.6(8); Lys, 4.0(4); Trp, 1.0(1). ##STR77## Physical Data

m.p. 155°-210° C. α!_(D) ²⁵ -46.15 (c 0.12, H₂ O)

FAB (C₁₉₅ H₃₃₄ N₆₂ O₅₈): M+H!⁺ 4475.8

AAA: Asx, 2.2(2); Glx, 6.9(7); Ser, 1.7(2); His, 3.2(3); Gly, 1.1(1);Ala, 3.1(3); Arg, 4.0(4); Thr, 0.9(1); Val, 1.1(1); Ile, 1.9(2); Leu,8.1(8); Lys, 4.1(4). ##STR78## Physical Data

m.p. 186°-218° C. α!_(D) ²⁵ -52.73 (c 0.265, H₂ O)

FAB(C₁₉₂ H₃₂₉ N₆₁ O ₅₇): M+H!⁺ 4404.4

AAA: Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.4(3); Gly, 1.1(1);Ala, 2.0(2); Arg, 3.8(4); Thr, 1.0(1); Val, 1.1(1); Ile, 1.7(2); Leu,7.9(8); Lys, 4.0(4). ##STR79## Physical Data

m.p. 169°-205° C. α!_(D) ²⁵ -50.78 (c 0.51, H₂ O)

FAB(C₁₈₆ H₃₁₇ N₅₇ O₅₆): M+H!⁺ 4248.0

AAA: Asx, 2.2(2); Glx, 6.8(7); Ser, 1.8(2); His, 3.3(3); Gly, 1.0(1);Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.0(1); Ile, 1.8(2); Leu,7.8(8); Lys, 3.6(4). ##STR80## Physical Data

m.p. 199°-205° C. α!_(D) ²⁵ -52.47 (c 0.41, H₂ O)

FAB(C₁₈₀ H₃₀₆ N₅₆ O₅₅): M+H!⁺ 4135.0

AAA: Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.3(3); Gly, 1.1(1);Ala, 2.0(2); Arg, 2.9(3); Thr, 1.0(1); Val, 1.1(1); Ile, 1.0(1); Leu,8.2(8); Lys, 3.8(4). ##STR81## Physical Data

m.p. 134.2° C. α!_(D) ²⁵ -48.12(c 0.36, H₂ O)

FAB(C₁₆₇ H₂₈₆ N₅₄ O₄₉): M+H!⁺ 3834.4

AAA: Asx, 2.0(2); Glx, 5.7(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.0(1);Thr, 0.9(1); Ala, 1.0(1); Arg, 2.8(3); Ile, 0.9(1); Leu, 7.4(8); Lys,4.3(4). ##STR82## Physical Data

m.p. 128.5-184.5° C. α!_(D) ²⁵ -6.53 (c 0.69, MeOH)

FAB(C₁₄₈ H₂₅₉ N₄₇ O₄₁): M+H!⁺ 3353

AAA: Asx, 2.0(2); Glx, 4.1(4); Ser, 0.9(1); His, 2.1(2); Gly, 1.0(1);Thr, 0.9(1); Ala, 1.0(1); Arg, 3.0(3); Ile, 1.0(1); Leu, 8.1(8); Lys,4.2(4). ##STR83## Physical Data

m.p. 165°-210° C. α!_(D) ²⁵ -36.05(c 0.12, H₂ O)

FAB (C₁₄₂ H₂₄₈ N₄₆ O₄₁): M+H!⁺ 3239.0

AAA: Asx, 2.0(2); Glx, 3.9(4); Ser, 0.9(1); His, 1.9(2); Gly, 1.0(1);Thr, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Ile, 0.9(1); Leu, 6.8(7); Lys,4.2(4). ##STR84## Physical Data

m.p. 150°-210° C. α!_(D) ²⁵ -18.0 (c 0.64, H₂ O)

FAB(C₂₀₈ H₃₅₁ N₇₉ O₆₀): M+H!⁺ 4918.6

AAA: Asx, 2.2(2); Glx, 6.2(6); Ser, 2.8(3); His, 3.1(3); Gly, 2.2(2);Thr, 2.2(2); Ala, 2.2(2); Arg, 10.4(10); Ile, 1.0(1); Leu, 8.0(8); Trp,1.1(1). ##STR85## Physical Data

m.p. 150°-210° C. α!_(D) ²⁵ -41.70(c 0.36, H₂ O)

FAB(C₁₈₉ H₃₂₄ N₇₂ O₅₂): M+H!⁺ 4437.14

AAA: Asx, 2.1(2); Glx, 4.1(4); Ser, 1.9(2); His, 2.0(2); Gly, 2.1(2);Thr, 2.0(2); Ala, 2.1(2); Arg, 9.7(10); Ile, 0.9(1); Leu, 7.4(8).

The side chain cyclized analog (Compound 57) was synthesized as aboveexcept the N.sup.α -Boc-N.sup.ε -Fmoc-Lys and N.sup.α -Boc-N.sup.γ-Fmoc-Asp were placed in positions 13 and 17, respectively. Uponcompletion of the Boc amino acid synthesis, the resin was traated with20% piperidine in DMF at room temperature for 30 minutes. The resin wasfiltered and washed with DMF, MeOH and CH₂ Cl₂. The resin (1.1 g) wassuspended in 10 ml DMF containing 250 mg PyBOP. The pH was adjusted to8-9 with DIEA and the resin stirred for 1 hour. The resin was filtered,washed with DMF and CH₂ Cl₂, then resuspended in DMF. The coupling wasrepeated using 125 mg of PyBop. The resin was filtered, washed with DMF,MeOH, and CH₂ Cl₂ and dried. The resin was then treated with HF and thepeptide purified as mentioned above. ##STR86## Physical Data

m.p. 142.5°-163.5° C. α!_(D) ²⁵ -34.31 (c 0.17, H₂ O)

FAB(C₁₇₅ H₂₉₈ N₅₆ O₅₀): M+H!+3986.4

AAA: Asx, 1.9(2); Glx, 5.9(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1);Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu,8.0(8); Lys, 4.0(4).

EXAMPLE II

Met³⁴, Ala³⁵ ! Compound 1, AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTMA-NH₂, (SEQID NO:25), was prepared and purified following the procedures of ExampleI. This polypeptide was converted to the homoserine lactone as follows.The purified peptide (160 mgs) was dissolved in 44% formic acid (4 mL).This solution was combined with a premixed solution of cyanogen bromide(700 mgs) and phenol (1.6 mgs) in 44% formic acid (4 mL) at 0° C. Thesolution was stirred at 0° C. for 2 hr and at room temperature for 2hrs. The formation of the product was monitored by HPLC (Vydac® C-18,300 Å, 4.6×250 mm, flow of 1.2 mL/min, gradient 25-45% acetonitrile in0.1% TFA over 10 min). The reaction was complete within 4 hr. Half ofthe sample was concentrated and purified by preparative RP-HPLC (Vydac®C-18, gradient 25-45% acetonitrile in 0.1% TFA). The homoserine lactonepeptide fractions were pooled and lyophilized to give 28 mgs of whitesolid of >95% purity, Compound 4. ##STR87## Physical Data

m.p. 138°-142° C. α!_(D) ²⁵ -50.66° (c 0.1, H₂ O)

FAB (C₁₇₆ H₂₉₉ N₅₅ O₅₂): M+H!⁺ 4017.61

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.0(3); Thi, 1.1(1);Ala, 1.1(1); Arg, 2.7(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys,3.8(4); Gly 1.09(1); hSer, 1.09(1).

Similarly, Compound 65 was prepared in accordance with this procedure.##STR88## Physical Data

m.p. 166°-176° C. α!_(D) ²⁵ -52.22 (c 0.25, H₂ O)

FAB(C180H₂₈₈ N₅₄ O₅₀) M+H!⁺ 4008.6

AAA: Asx, 3.1(3); Glx, 4.8(5); Ser, 2.9(3); His, 2.9(3); Gly, 1.1(1);Ala, 1.1(1); Arg, 2.0(2); Val, 2.7(3); Phe, 1.0(1); Ile, 1.0(1); Leu+Nle 5.9 (4+2); Lys, 2.8(3).

EXAMPLE III

To prepare the homoserine amide, the crude hSerlactone analog, Compound4, was concentrated and treated with 25 mL saturated NH₃ in methanol.The solution was stirred at 0° C. for 2 hr and at room temperature for16 hr. The reaction was monitored by HPLC (Vydac® C-18, 300 Å, 4.6×250mm, flow of 1.2 mL/min, gradient 20-45% acetonitrile in 0.1% TFA) andwas complete within 18 hr. The solution was concentrated and purified bypreparative RP-HPLC (Vydac® C-18, gradient of 25-45% acetonitrile in0.1% TFA). The homoserine amide peptide fractions were pooled andlyophilized to give 30 mgs of white solid of >98% purity, Compound 3.##STR89## Physical Data

m.p. 138°-142° C. α!_(D) ²⁵ -45.97° (c 0.25, H₂ O)

FAB (C₁₇₆ H₃₀₂ N₅₆ O₅₂): M+H!⁺ 4033.9

AAA: Asp, 2.1(2); Glu, 6.1(6); Ser, 1.6(2); His, 2.8(3); Gly, 0.97(1);hSer, 0.97(1); Thi, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Val, 1.0(1); Ile,1.0(1); Leu, 7.6(8); Lys, 3.9(4).

Similarly, Compounds 22, 23 and 28 were prepared following thisprocedure. ##STR90## Physical Data

m.p. 69.4°-128° C. α!_(D) ²⁵ -43.93 (c 0.15, H₂ O)

FAB(C₁₇₉ H₃₀₂ N₅₆ O₄₉): M+H!⁺ 4022.9

AAA: Asx, 2.0(2); Glx, 4.9(5); Ser, 2.6(3); His, 2.8(3); Gly, 1.0(1);Ala, 1.0(1); Arg, 3.0(3); Val, 1.0(1); Phe, 1.0(1); Ile, 0.9(1); Leu,8.8(9); Lys, 3.4(3); ##STR91## Physical Data

m.p. 87.1°-142.1° C. α!_(D) ²⁵ -52.14 (c 0.41, H₂ O)

FAB (C₁₇₉ H₃₀₃ N₅₇ O₄₉): M+H!⁺ 4038

AAA: Asx, 2.1(2); Glx, 4.9(5); Ser, 2.7(3); His, 2.8(3); Gly, 1.0(1);hSer, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Phe, 0.9(1); Ile,0.9(1); Leu, 7.9(8); Lys, 3.7(4). ##STR92## Physical Data

m.p. 80° C. α!_(D) ²⁵ -48.64 (c 0.09, H₂ O)

FAB (C₁₉₃ H₃₃₄ N₇₀ O₅₆): M+H!⁺ 4530.0

AAA: Asx, 2.2(2); Glx, 6.1(6); Ser, 1.7(2); His, 3.0(3); Gly, 1.9(2);hSer, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 7.2(7); Val, 0.8(1); Ile,1.0(1); Leu, 8.4(8); Lys, 2.1(2).

The homoserine alkylamides were similarly prepared from the homoserinelactone by dissolving it in DMF containing an excess of thecorresponding alkylamine. After stirring at room temperature for severaldays (the reaction was monitored by analytical HPLC) the mixture wasevaporated to dryness and the peptide purified by preparative HPLC.Representative homoserine alkylamides are Compounds 55 and 56. ##STR93##Physical Data

m.p. (not determined) α!_(D) ²⁵ (not determined)

FAB (C₁₇₈ H₃₀₆ N₅₆ O₅₂): M+H!⁺ 4063.0

AAA: Asx, 2.1(2); Glx, 5.8(6); Ser, 1.7(2); His, 3.1(3); Gly, 0.9(1);Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu,8.4(8); Lys, 3.7(4); hSer, 0.9(1). ##STR94## Physical Data

m.p. (not determined) α!D²⁵ (not determined)

FAB (C₁₈₄ H₃₁₀ N₅₆ O₅₂): M+H!⁺ 4138.8

AAA: Asx, 2.0(2); Glx, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 0.9(1);Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3); Val, 1.0(1); Ile, 0.9(1); Leu,8.0(8); Lys, 4.1(4); hSer, 0.9(1).

EXAMPLE IV

The cesium salt of N.sup.α -Boc-N.sup.γ -Fmoc-L-2,4-diaminobutyric acidwas attached to Merrifield resin (DMF, 50° C., 48 hrs) and used in asolid-phase synthesis in place of the Boc-Ala resin as in Example I.Upon completion of the synthesis the peptide was treated with 20%piperidine in DMF at room temperature for 30 minutes to remove the sidechain Fmoc protecting group. The protected peptide spontaneouslycyclized to the lactam thereby cleaving itself from the resin. Thesolution was filtered from the resin and evaporated under vacuum toleave on oil. The residue was treated with liquid HF as in Example I toyield the crude unprotected peptide. The peptide was treated andpurified as in Example I. ##STR95## Physical Data

m.p. 161°-181° C. α!_(D) ²⁵ -48.38 (c 0.25, H₂ O)

FAB(C₁₇₆ H₃₀₀ N₅₆ O₅₁): M+H!⁺ 4016.8

AAA: Asx, 2.1(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.3(3); Gly, 1.1(1);Thr, 1.0(1); Ala, 2.1(2); Arg, 2.9(3); Val, 0.9(1); Ile, 0.9(1); Leu,8.0(8); Lys, 3.8(4).

EXAMPLE V

An aqueous solution of the homoserine lactone analog from Example II wastreated with porcine liver esterase (Sigma Chemical Company, St. Louis,Mo.). The hydrolysis of the lactone to the C-terminal homoserine wasmonitroed by analytical HPLC. When the hydrolysis was judged to becomplete the material was pruified by preparative HPLC as in Example I.##STR96## Physical Data

m.p. (not determined) α!_(D) ²⁵ (not determined)

FAB (C₁₇₆ H₃₀₁ N₅₅ O₅₃): M+H!+4035.1

AAA: Asx, 2.1(2); Glx, 5.9(6); Ser, 2.0(2); His, 3.1(3); Gly, 0.8(1);hSer, 0.8(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.3(1); Ile,1.0(1); Leu, 8.1(8); Lys, 3.8(4).

EXAMPLE VI

Following Example I, the protected peptide-resinBocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD(OBz)LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)AOPAMwas synthesized on a 0.35 mmol scale. All N.sup.α groups were protectedwith t-butoxycarbonyl (Boc); side chain protecting groups were asindicated. After completion of the synthesis, the peptide resin wastreated with 50 mL of 20% piperidine in dimethylformamide (DMF) at roomtemperature for 30 minutes to remove the fluorenylmethoxycarbonyl (Fmoc)protecting group on lysine. The resin was washed successively with DMF,MeOH, CH₂ Cl₂ and dried to give 1.6 g partially protected peptide. 0.8 g(0.175 mmol) of the partially protected peptide was acylated on lysinewith 0.44 g (0.3 mmol) of methoxydi(ethyleneoxy) acetic acid PEG(2)CH₂COOH! in the presence of 0.16 g (0.3 mmol)benzotriazolyloxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBop) and 0.067 g (0.525 mmol) of diisopropylethyl amine (DIEA) in 20mL DMF at room temperature for 5 hrs. After 5 hrs., the resin wasfiltered and washed successively with DMF, MeOH and CH₂ Cl₂. Theacylation step was repeated twice until negative ninhydrin result on theresin was obtained. The final peptide was cleaved from the resin withremoval of the side chain protecting groups and purification as inExample I; 100 mgs of Compound 19 were obtained. ##STR97## Physical Data

m.p. 145°-195° C. α!_(D) ²⁵ -44.60° (c 0.2, H₂ O)

FAB (C₁₈₃ H₃₁₆ N₆₄ O₅₄): M+H!+4276.2

AAA: Asp, 2.1(2); Glu, 5.0(5); Ser, 1.6(2); His, 2.9(3); Gly, 0.9(1);Thr, 1.9(2); Arg, 7.1(7); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys,0.9(1).

EXAMPLE VII

Peptide was synthesized, cleaved and purified in the same manner as inExample IV except 2-methoxypoly (ethyleneoxy) acetic acid PEG(5000)CH₂CO₂ H! was used as the acylating agent. 0.8 g (0.175 mmol) of partiallyprotected peptide yielded 300 mgs of pure Compound 20. ##STR98##Physical Data

m.p. 105° C. α!_(D) ²⁵ -22.95° (c 0.11, 50% aq. HOAc)

AAA: Asp, 2.0(2); Glu, 4.8(5); Ser, 1.6(2); His, 2.6(3); Gly, 1.1(1);Thr, 1.1(1); Arg, 7.3(7); Val, 0.8(1); Ile, 0.9(1); Leu, 8.3(8); Lys,1.1(1); Ala, 1.8(2).

EXAMPLE VIII Synthesis of hPTHrp(1-34) analog gene

A synthetic gene coding for the hPTHrp(1-34) analog Compound 4 (SEQ IDNO:9) was designed having the nucleotide sequence and enzyme restrictionsites shown in FIG. 1. The requisite oligodeoxynucleotides were preparedwith a DNA synthesizer (Milligen/Biosearch) using the phosphoramiditeprocess of Sinha, et al., Nucleic Acid Research 12, 4539-4557 (1984).After deprotection, the crude oligonucleotides were purified by gelelectrophoresis on preparative 15% polyacrylamide gels. Theoligonucleotides were located with UV, excised from the gel, desaltedover Waters c18 Sep-pak® cartridges, and lyophilized.

Amplification via polymerase chain reaction (PCR) was carried out on aPerkin-Elmer Cetus thermal cycler, with 25 cycles of: 94° C. for 1minute, 50° C. for 2 minutes, and 72° C. for 3 minutes, using reagents,including Taq polymerase, from the "GeneAmp" DNA amplification kit(Perkin-Elmer Cetus)

Two overlapping oligonucleotides, an 88 mer (2 μg), PTH3, ##STR99## wereprepared as the template DNA sequence for the hPTHrp (1-34) analog gene.Utilizing the two flanking primers, PTHPCR1: CCTCTAGATC TCCGCGGCGC TAG(SEQ ID NO:33) and PTHPCR2: CCTCGAAGCT TATGCATCAT TATC (SEQ ID NO:34),the entire gene was amplified by PCR. The amplified DNA products werepurified by gel electrophoresis on 4% NuSieve® agarose gel. The bandcontaining the synthetic hPTHrp(1-34) analog gene, approximately 150bases in length, was excised from the gel and approximately 200 ng ofDNA isolated by Elu-Quik® glass gel DNA extraction (Schleicher &Schuell, Keene, N.H.).

EXAMPLE IX Molecular Cloning of an hPTHrp(1-34)1 Analog Gene

To subclone the hPTHrp(1-34) analog gene of Example VI, 200 ng of theamplified DNA was isolated and cut by restriction enzymes HinD III andSac II. As shown in FIG. 2, the DNA was ligated to 2 μg TrpLE 18 Prot(Ile³,Pro⁵) plasmid previously cleaved with Hind III and Sac II.

The resulting plasmid, TrpLE 18 hPTHrp(1-34)1, containing one copy ofthe hPTHrp(1-34) analog gene was then transformed into competent E. coliHB 101 cells (CLONTECH, Palo Alto, Calif.). Transformants were subjectedto PCR analysis to verify insertion. Transformed cell colonies wereselected and boiled in 200 μL of water for 5 minutes; 2 μL weresubjected to PCR with two primers flanking the insert. The PCR productwas then analyzed on 1% agarose gel to confirm the presence of one copyof the hPTHrp(1-34) gene insert. TrpLE 18 hPTHrp(1-34)1 construct wasthen verified by DNA sequencing on an automated DNA sequencer (AppliedBiosystems Model 373A, Foster City, Calif.) using the vendor's Dye DeoxyTerminator Sequencing kit.

EXAMPLE X Construction of a Trp LE 18 vector containing multiple copiesof the hPTHrp(1-34) analog gene

Unique Nhe I and Xba I restriction sites are located near the beginningand end of the hPTHrp(1-34) analog gene sequence. These two sites, whichrecognize different sequences, but produce identical single strandcohesive termini, allow the construction of multiple copy hPTHrp (1-34)genes within the Trp LE 18 vector.

The strategy for constructing repeated hPTHrp(1-34) sequences in tandemis outlined in FIG. 3. In separate reactions, 5 μg of plasmid Trp LE 18hPTHrp(1-34)1 containing a single copy of the gene was cleaved with BamHI+Nhe I and Xba I+Bam HI. From each digest, about 300 ng of thefragment containing the hPTHrp(1-34) analog gene was isolated. These twofragments were mixed and ligated to form the Trp LE 18 hPTHrp(1-34)2plasmid. This plasmid was used to transform competent E. coli HB 101cells. Sizing of the transformed PCR products on 1% agarose gel was usedto determine the presence of two copies of the hPTHrp(1-34) gene insert.TrpLE 18 hPTHrp(1-34)2 containing two copies of the gene was thenconfirmed by DNA sequencing. The correct fusion of the two hPTHrp(1-34)genes results in the elimination of Nhe I and Xba I sites at thejunction. This makes the remaining Xba I and Nhe I sites flanking thetandem genes unique.

By repeating this process the final plasmid Trp LE 18 hPTHrp(1-34)4containing four copies of the hPTHrp(1-34) gene was constructed, asshown in FIG. 4. The sequence of Trp LE 18 hPTHrp(1-34)4 was found to becorrect by DNA sequence analysis.

EXAMPLE XI Expression and Purification of Trp LE 18 hPTHrp(1-34)4

Induction of the Trp LE 18 hPTHrp(1-34)4.

A starter culture of 50 mL of LB culture media, J. H. Miller,"Experiments in Molecular Genetics," p.431 (1972), incorporated hereinby reference, containing 50 μg/mL ampicillin and 100 μg/mL tryptophan,was inoculated with E. coli cells containing Trp LE 18 hPTHrp(1-34)4plasmid, and grown overnight at 37° C. with vigorous shaking to an A₅₅₀of about 6. Two liters of LB culture media for production werepre-warmed to 37° C. and seeded with 20 mL of the starter culture togive an A₅₅₀ of about 0.06. The culture was then grown with vigorousshaking to an A₅₅₀ of between 0.6 and 0.8, whereupon 2 mL of a 10 mg/mLsolution of indole acrylic acid (IAA) was added. Growth was continuedwith good aeration for about 16 hr to a final A₅₅₀ of about 6(typically, between 4 and 10). The cells were concentrated bycentrifugation and resuspended in 500 mL of 50 mM Tris-HCl, pH 7.5, 0.1mM EDTA buffer solution (Tris buffer).

The suspension was sonicated using a Heat Systems-Ultrasonics, Inc.model 220F sonicator (equipped with a 3/4" horn) operated at 50% of fullcapacity to avoid overheating.

To determine the extent of induction, the whole cells were analyzed bySDS-PAGE. The gene products derived from the TrpLE 18 hPTHrp(1-34)4construct were seen as a major band of the predicted MW of approximately17,000. This accounts for as much as 10% of the total cellular protein.

Isolation of the Fusion Protein.

The cell lysate was centrifuged for 15 min. at about 3600×g to pelletthe Trp LE 18 hPTHrp(1-34)4 fusion protein; the supernatant wasdiscarded. The pellet was resuspended in 200 mL Tris buffer. (typically40-80 A_(550/) mL).

EXAMPLE XII Processing of the Fusion Protein and Purification ofhomo-Serlactone hPTHrp(1-34) peptide

Cleavage of the methionine residues flanking the hPTHrp(1-34) multimericfusion protein with CNBr releases the desired homo-SerlactonehPTHrp(1-34) polypeptide, which was purified as described below.

CNBr Treatment of Fusion Protein.

The washed pellet of TrpLE 18 hPTHrp(1-34)4 fusion protein wasresuspended by gently stirring in 60 mL of 70% formic acid (about 20mg/mL total protein; typically material from 1000 A₅₅₀ units of cells isdissolved in 3 mL). A few drops of octanol were added and N₂ bubbledthrough the solution for 20 minutes before adding 5.5 g CNBr. Thisreaction was allowed to proceed for 6 hours at 25° C. before an equalvolume of 50:50 MeOH:H₂ O was mixed with the sample and subsequentlyremoved by rotary evaporation. After 2 to 4 repetitions of this process,the bulk of the formic acid and CNBr were essentially removed. Thesample was then evaporated to dryness, redissolved in 200 mL water andlyophilized for storage.

Purification of homo-Serlactone hPTHrp(1-34).

The CNBr cleaved supernatant was dialyzed against 50 mM KH₂ PO₄ pH 6.5for 24 hours with multiple changes. During dialysis, pH was maintainedat 6.5. After dialysis, the precipitates were removed by high speedcentrifugation. The supernatant was clarified through a Gelman 0.45μfilter device (Acrodisc 4184).

Cation Exchange Chromatography.

Initial purification was accomplished by cation exchange chromatographyon a Bio-Gel TSK-SP-5PW HPLC column (21.5×150 mm). Chromatographicconditions for a flow rate of 8 mL/min and a yield of approximately 12mg of highly purified homo-Serlactone hPTHrp(1-34) peptide were:

1. Column equilibration in 50 mM KH₂ PO₄, pH 6.5

2. Load 10 mL clarified supernatant (approximately 1.5 L culture brothor 2.4 g inclusion).

3. Wash column with 50 mM KH₂ PO₄ pH 6.5 containing 50 mM NaCl untilbaseline is stabilized.

4. Elute column with 50 mM KH₂ PO₄ pH 6.5 containing 90 mM NaCl. Collectfractions for about 45 minutes.

5. Analyze the 90 mM NaCl fractions for homoSerlactone hPTHrp(1-34)content by C18 HPLC before pooling and storage.

Reverse Phase HPLC Chromatography.

A reverse phase Poros R/H 4.6×100 mm column (Perseptive Biosystems,Cambridge, Mass.) was used for the final purification step. Thechromatographic conditions were as follows:

    ______________________________________                                        Mobile phase A:                                                                            0.1% trifluoroacetic acid (TFA)/water                            B:           0.1% trifluoroacetic acid (TFA)/CH.sub.3 CN                      TIME            FLOW     % B                                                  ______________________________________                                        0 min           4 ml/min 15                                                   5.0 min         4 ml/min 40                                                   5.2 min         4 ml/min 100                                                  6.8 min         4 ml/min 100                                                  7.0 min         4 ml/min 15                                                   ______________________________________                                    

Retention time of the homo-Serlactone hPTHrp(1-34), Compound 4, wasapproximately 2.943 minutes. The purified peptide was approximately 98%pure as determined by mass spectroscopy.

EXAMPLE XIII

The compounds of this invention were evaluated for their effect on bonemass in ovariectomized rats, generally in accord with the procedures ofGunness-Hey and Hock, Metab. Bone Dis. 5:177 181 (1984).

Adult Sprague-Dawley female rats were acclimatized, weight grouped (n=9,10 or 12), and subjected to bilateral ovariectomy (OVX) or sham surgery.Dosing was initiated 17 days after surgery and continued for 20 days.Test compound was administered subcutaneously once a day in 2% ratserum/saline vehicle.

After 20 days of dosing, the rats were sacrificed and the right femursexcised. The femurs were cut in half and the distal half femurs (DHF)were further separated into trabecular bone (TB) and cortical bone (CB)by drilling out the trabeculae. Calcium was extracted and measured byCalcette calcium analyzer and expressed as mean bone Ca in mg/DHF/100 gbody weight.

The two sample t-test was used to compare OVX and sham groups. One-wayANOVA was used to compare OVX groups, followed by Fisher's LSD multiplecomparison to compare each treatment group to vehicle.

Ovariectomy induced substantial total bone loss, primarily fromtrabecular bone. Total bone calcium was 47 to 54% lower than forsham-operated controls.

bPTH(1-34) and hPTHrp(1-34) at 80 μg/kg/day provided statisticallysignificant increases in total bone calcium for treated OVX rats,ranging from 53 to 95% and 18 to 40%, respectively; however, there wasno significant increase in cortical bone calcium.

Compounds of this invention, dosed at 80 μg/kg/day, increased total bonecalcium by from 66 to 138% and trabecular calcium by from 87 to 128%.Cortical bone calcium, trabecular thickness, and bone volume were alsosignificantly increased over untreated OVX controls.

In this assay, the following compounds were tested:

    ______________________________________                                                             Trabecular Bone                                                                             Total Bone                                                      Calcium       Calcium                                    Compound    n        (% increase   (% increase                                Number      (# of tests)                                                                           over OVX)     over OVX)                                  ______________________________________                                        Compound 1  6        101-128%      88-138%                                    (SEQ ID NO: 7)                                                                Compound 2  3         87-102%      66-114%                                    (SEQ ID NO: 6)                                                                Compound 4  3        --            88-114%                                    (SEQ ID NO: 9)                                                                ______________________________________                                    

In similar studies, ovariectomized rats were dosed for 5, 10 and 20days, at 40 ug/kg/day, with the following results:

    ______________________________________                                                                           Total Bone                                                                    Calcium                                    Compound       n        # of Days (d)                                                                            (% increase                                Number         (# of tests)                                                                           of dosing  over OVX)                                  ______________________________________                                        Compound 1 (SEQ ID NO: 7)                                                                    3        20d        73-109%                                    Compound 4 (SEQ ID NO: 9)                                                                    5        20d        79-105%                                    Compound 4 (SEQ ID NO: 9)                                                                    1        10d        79%                                        Compound 49    1        10d        93%                                        (SEQ ID NO: 63)                                                               Compound 4 (SEQ ID NO: 9)                                                                    1         5d        55%                                        Compound 42    1         5d        60%                                        (SEQ ID NO: 56)                                                               ______________________________________                                    

EXAMPLE XIV TOXICITY

In the above Example XI, no toxic effects were observed with thecompounds of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 86                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuAsn                              151015                                                                        SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaValSerGluIleGlnPheMetHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerMetGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       SerValSerGluIleGlnLeuMetHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerLeuGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnPhe                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgPhePheLeuHisHisLeuIleAlaGluIleHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuArgLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa34 =homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa34 =homoserine lactone"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGlyArgArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuLys                              202530                                                                        GluLeu                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuAlaArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgAlaGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AlaValSerGluAlaGlnLeuLeuHisAspLeuGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AlaLeu                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuLysArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note="Xaa29 =                                         lysine- (OCCH2(OCH2CH2)2OCH3)"                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note="Xaa29 =                                         lysine- (OCCH2(OCH2CH2)110OCH3"                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuXaaArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgAlaLeuAlaGluAlaLeuAlaGluAlaLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgSerLeuLeuSerSerLeuLeuSerSerLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgAlaPheTyrAspLysValAlaGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =norleucine"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note="Xaa18 =norleucine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerXaaGluArgValGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =norleucine"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note="Xaa18 =norleucine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AlaValSerGluIleGlnPheXaaHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerXaaArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrMetAla                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =glutamic acid or                          arginine"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="Sequence 26 is embedded at                      positions 22 to 31 of sequences 5, 6, 7, 8, 9, 10,                            11, 12, 13, and 14."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GluLeuLeuGluLysLeuLeuXaaLysLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =glutamic acid,                            lysine, or lysine-(OCCH2PEGX)"                                                (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="Sequence 27 is embedded at                      positions 22 to 31 of sequences 15, 16, 17, 18,                               and 19. "                                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GluLeuLeuGluArgLeuLeuXaaArgLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="Sequence 28 is embedded at                      positions 22 to 31 of sequence 20."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AlaLeuAlaGluAlaLeuAlaGluAlaLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="Sequence 29 is embedded at                      positions 22 to 31 of sequence 21."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      SerLeuLeuSerSerLeuLeuSerSerLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="Sequence 30 is embedded at                      positions 22 to 31 of sequence 22."                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AlaPheTyrAspLysValAlaGluLysLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CCTCTAGATCTCCGCGGCGCTAGCATGGCTGTTTCTGAACATCAGCTGCTTCATGACAAA60                GGTAAATCGATTCAAGATCTGAGACGTC88                                                (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CCTCGAAGCTTATGCATCATTATCTAGACATAGTATGCAGCTTTTCAAGCAGTTTCTCCA60                GCAGCTCGCGACGTCTCAGATCTTGAATCG90                                              (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CCTCTAGATCTCCGCGCGCTAGC23                                                     (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CCTCGAAGCTTATGCATCATTATC24                                                    (2) INFORMATION FOR SEQ ID NO: 35:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:                                     AlaValSerGluIleGlnPheLeuHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO: 36:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:                                     AlaValSerGluIleGlnPheLeuHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 37:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:                                     AlaValSerGluIleGlnPheLeuHisAsnLysGlyLysHisLeuSer                              151015                                                                        SerLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        AsnXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 38:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuLysLeuLysGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 39:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 40:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAlaPro                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 41:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAlaGlyArgArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 42:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 38                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAlaGlyArgArgXaa                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO: 43:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrTyr                                                                        (2) INFORMATION FOR SEQ ID NO: 44:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyTyrSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 45:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyCysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 46:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /note="Xaa is                                          Cys(CH- 2CONH(CH-2)-2NH(biotinyl))"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyXaaSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 47:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /note="Xaa is                                          Lys(7- dimethylamino-2-oxo-2H-1-benxopyran-4-acety                            l)"                                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyXaaSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 48:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGly                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 49:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note="Xaa4 is Glu(OCH-3)"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note="Xaa10 is Asp(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa17 is Asp(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 22                                                              (D) OTHER INFORMATION: /note="Xaa22 is Glu(OCH3)"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 25                                                              (D) OTHER INFORMATION: /note="Xaa25 is Glu(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note="Xaa29 is Glu(OCH-3)"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:                                     AlaValSerXaaHisGlnLeuLeuHisXaaLysGlyLysSerIleGln                              151015                                                                        XaaLeuArgArgArgXaaLeuLeuXaaLysLeuLeuXaaLysLeuHis                              202530                                                                        Ala                                                                           (2) INFORMATION FOR SEQ ID NO: 50:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /note="Xaa4 is Glu(OCH-3)"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /note="Xaa10 is Asp(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 17                                                              (D) OTHER INFORMATION: /note="Xaa17 is Asp(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 22                                                              (D) OTHER INFORMATION: /note="Xaa22 is Glu(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 25                                                              (D) OTHER INFORMATION: /note="Xaa25 is Glu(OCH-3)"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 29                                                              (D) OTHER INFORMATION: /note="Xaa29 is Glu(OCH-3)"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:                                     AlaValSerXaaHisGlnLeuLeuHisXaaLysGlyLysSerIleGln                              151015                                                                        XaaLeuArgArgArgXaaLeuLeuXaaLysLeuLeuXaaLysLeuHis                              202530                                                                        Ala                                                                           (2) INFORMATION FOR SEQ ID NO: 51:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 52:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaPro                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 53:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrPro                                                                        (2) INFORMATION FOR SEQ ID NO: 54:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrPro                                                                        (2) INFORMATION FOR SEQ ID NO: 55:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        Pro                                                                           (2) INFORMATION FOR SEQ ID NO: 56:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuPro                              202530                                                                        (2) INFORMATION FOR SEQ ID NO: 57:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrArgSerAlaTrp                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 58:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:                                     AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrAlaGlyArgArgThrArgSerAlaTrp                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 59:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:                                     AlaValSerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrArgGlyArgArgThrArgSerAlaTrp                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 60:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 13                                                              (D) OTHER INFORMATION: /note="Xaa13 is                                        Lys(dihydrocinnamoyl)"                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:                                     AlaValSerGluHisGlnLeuLeuHisAspArgGlyXaaSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHis                              202530                                                                        ThrArgGlyArgArgThrArgSerAlaTrp                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 61:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Leu8 is Norleucine"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note="Leu18 is Norleucine"                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:                                     AlaValSerGluIleGlnPheLeuHisAsnLeuGlyLysHisLeuSer                              151015                                                                        SerLeuThrArgSerAlaTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnTyr                                                                        (2) INFORMATION FOR SEQ ID NO: 62:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrMetAla                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 63:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 33                                                              (D) OTHER INFORMATION: /note="Xaa is Thr                                      1,4- diaminobutyryl lactam"                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        Xaa                                                                           (2) INFORMATION FOR SEQ ID NO: 64:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgPhePheLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 65:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuHisLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 66:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluHisLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 67:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuIleAlaLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 68:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluGluIleHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 69:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 70:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine"                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 71:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAla                                                                        (2) INFORMATION FOR SEQ ID NO: 72:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrArgSerAlaTrp                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 73:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 36                                                              (D) OTHER INFORMATION: /note="Xaa is Ala                                      3-(2- naphthyl)-L-alanine"                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrArgSerXaa                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 74:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaSerAlaTrp                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 75:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGluIleArgAla                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO: 76:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGluIleArg                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO: 77:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGluIle                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO: 78:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:                                     AlaValSerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGln                              151015                                                                        AspLeuArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHis                              202530                                                                        ThrAlaGlu                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO: 79:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: N-terminal                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Leu8 is Norleucine"                             (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 18                                                              (D) OTHER INFORMATION: /note="Leu18 is Norleucine"                            (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 34                                                              (D) OTHER INFORMATION: /note="Xaa is homoserine lactone"                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:                                     AlaValSerGluIleGlnPheLeuHisAsnLysGlyLysHisLeuSer                              151015                                                                        SerLeuGluArgValGluTrpLeuArgLysLysLeuGlnAspValHis                              202530                                                                        AsnXaa                                                                        (2) INFORMATION FOR SEQ ID NO: 80:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:                                     SerGluHisGlnLeuLeuHisAspLysGlyLysSerIleGlnAspLeu                              151015                                                                        ArgArgArgGluLeuLeuGluLysLeuLeuGluLysLeuHisThrAla                              202530                                                                        (2) INFORMATION FOR SEQ ID NO: 81:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:                                     LeuLeuHisAspLysGlyLysSerIleGlnAspLeuArgArgArgGlu                              151015                                                                        LeuLeuGluLysLeuLeuGluLysLeuHisThrAla                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO: 82:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:                                     LeuHisAspLysGlyLysSerIleGlnAspLeuArgArgArgGluLeu                              151015                                                                        LeuGluLysLeuLeuGluLysLeuHisThrAla                                             2025                                                                          (2) INFORMATION FOR SEQ ID NO: 83:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:                                     SerGluHisGlnLeuLeuHisAspArgGlyArgSerIleGlnAspLeu                              151015                                                                        ArgArgArgGluLeuLeuGluArgLeuLeuGluArgLeuHisLeuHis                              202530                                                                        ArgGlyArgArgThrArgSerAlaTrp                                                   3540                                                                          (2) INFORMATION FOR SEQ ID NO: 84:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:                                     LeuLeuHisAspArgGlyArgSerIleGlnAspLeuArgArgArgGlu                              151015                                                                        LeuLeuGluArgLeuLeuGluArgLeuHisAlaGlyArgArgThrArg                              202530                                                                        SerAlaTrp                                                                     35                                                                            (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note="Xaa1 and Xaa4 =Glu,                             Glu(OCH3), His, or Phe"                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Xaa2 =Leu or Phe"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="Xaa5 =Lys or His"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7 and 10                                                        (D) OTHER INFORMATION: /note="Xaa7 and Xaa10 =                                Leu or Ile"                                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =Ala, Arg, or Glu"                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note="Xaa9 =Lys or Glu"                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      XaaXaaLeuXaaXaaLeuXaaXaaXaaXaa                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1 and 4                                                         (D) OTHER INFORMATION: /note="Xaa1 and Xaa4 =Glu,                             Glu(OCH3), His, or Phe"                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /note="Xaa2 =Leu or Phe"                               (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa8 =Glu, Lys or                               Lys(COCH2PEGX)"                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      XaaXaaLeuXaaArgLeuLeuXaaArgLeu                                                1510                                                                          __________________________________________________________________________

We claim:
 1. A process for the synthesis of a modified PTH or PTHrPhaving bone mass restoring activity which differs from naturallyoccurring PTHrP or PTHrP by changes comprising substitutions at one ormore of positions (22-31), wherein the substitutions are selected fromthe group consisting of SEQ ID Nos. (26, 27, 28, 29, 30, 85 and 86),said process comprising sequentially coupling protected amino acids,optionally on a resin, deprotecting, and purifying the polypeptideproduct.
 2. A process of claim 1 wherein the substitution comprises SEQID No:
 26. 3. A process of claim 1 wherein the substitution comprisesSEQ ID No:
 27. 4. A process of claim 1 wherein the substitutioncomprises SEQ ID No:
 28. 5. A process of claim 1 wherein thesubstitution comprises SEQ ID No:
 29. 6. A process of claim 1 whereinthe substitution comprises SEQ ID No:
 30. 7. A process of claim 1wherein the substitution comprises SEQ ID No:
 85. 8. A process of claim1 wherein the substitution comprises SEQ ID No: 86.